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Significance of the Study – Examples and Writing Guide

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Significance of the Study

Definition:

Significance of the study in research refers to the potential importance, relevance, or impact of the research findings. It outlines how the research contributes to the existing body of knowledge, what gaps it fills, or what new understanding it brings to a particular field of study.

In general, the significance of a study can be assessed based on several factors, including:

• Originality : The extent to which the study advances existing knowledge or introduces new ideas and perspectives.
• Practical relevance: The potential implications of the study for real-world situations, such as improving policy or practice.
• Theoretical contribution: The extent to which the study provides new insights or perspectives on theoretical concepts or frameworks.
• Methodological rigor : The extent to which the study employs appropriate and robust methods and techniques to generate reliable and valid data.
• Social or cultural impact : The potential impact of the study on society, culture, or public perception of a particular issue.

Types of Significance of the Study

The significance of the Study can be divided into the following types:

Theoretical Significance

Theoretical significance refers to the contribution that a study makes to the existing body of theories in a specific field. This could be by confirming, refuting, or adding nuance to a currently accepted theory, or by proposing an entirely new theory.

Practical Significance

Practical significance refers to the direct applicability and usefulness of the research findings in real-world contexts. Studies with practical significance often address real-life problems and offer potential solutions or strategies. For example, a study in the field of public health might identify a new intervention that significantly reduces the spread of a certain disease.

Significance for Future Research

This pertains to the potential of a study to inspire further research. A study might open up new areas of investigation, provide new research methodologies, or propose new hypotheses that need to be tested.

How to Write Significance of the Study

Here’s a guide to writing an effective “Significance of the Study” section in research paper, thesis, or dissertation:

• Background : Begin by giving some context about your study. This could include a brief introduction to your subject area, the current state of research in the field, and the specific problem or question your study addresses.
• Identify the Gap : Demonstrate that there’s a gap in the existing literature or knowledge that needs to be filled, which is where your study comes in. The gap could be a lack of research on a particular topic, differing results in existing studies, or a new problem that has arisen and hasn’t yet been studied.
• State the Purpose of Your Study : Clearly state the main objective of your research. You may want to state the purpose as a solution to the problem or gap you’ve previously identified.
• Contributes to the existing body of knowledge.
• Addresses a significant research gap.
• Offers a new or better solution to a problem.
• Impacts policy or practice.
• Leads to improvements in a particular field or sector.
• Identify Beneficiaries : Identify who will benefit from your study. This could include other researchers, practitioners in your field, policy-makers, communities, businesses, or others. Explain how your findings could be used and by whom.
• Future Implications : Discuss the implications of your study for future research. This could involve questions that are left open, new questions that have been raised, or potential future methodologies suggested by your study.

Significance of the Study in Research Paper

The Significance of the Study in a research paper refers to the importance or relevance of the research topic being investigated. It answers the question “Why is this research important?” and highlights the potential contributions and impacts of the study.

The significance of the study can be presented in the introduction or background section of a research paper. It typically includes the following components:

• Importance of the research problem: This describes why the research problem is worth investigating and how it relates to existing knowledge and theories.
• Potential benefits and implications: This explains the potential contributions and impacts of the research on theory, practice, policy, or society.
• Originality and novelty: This highlights how the research adds new insights, approaches, or methods to the existing body of knowledge.
• Scope and limitations: This outlines the boundaries and constraints of the research and clarifies what the study will and will not address.

Suppose a researcher is conducting a study on the “Effects of social media use on the mental health of adolescents”.

The significance of the study may be:

“The present study is significant because it addresses a pressing public health issue of the negative impact of social media use on adolescent mental health. Given the widespread use of social media among this age group, understanding the effects of social media on mental health is critical for developing effective prevention and intervention strategies. This study will contribute to the existing literature by examining the moderating factors that may affect the relationship between social media use and mental health outcomes. It will also shed light on the potential benefits and risks of social media use for adolescents and inform the development of evidence-based guidelines for promoting healthy social media use among this population. The limitations of this study include the use of self-reported measures and the cross-sectional design, which precludes causal inference.”

Significance of the Study In Thesis

The significance of the study in a thesis refers to the importance or relevance of the research topic and the potential impact of the study on the field of study or society as a whole. It explains why the research is worth doing and what contribution it will make to existing knowledge.

For example, the significance of a thesis on “Artificial Intelligence in Healthcare” could be:

• With the increasing availability of healthcare data and the development of advanced machine learning algorithms, AI has the potential to revolutionize the healthcare industry by improving diagnosis, treatment, and patient outcomes. Therefore, this thesis can contribute to the understanding of how AI can be applied in healthcare and how it can benefit patients and healthcare providers.
• AI in healthcare also raises ethical and social issues, such as privacy concerns, bias in algorithms, and the impact on healthcare jobs. By exploring these issues in the thesis, it can provide insights into the potential risks and benefits of AI in healthcare and inform policy decisions.
• Finally, the thesis can also advance the field of computer science by developing new AI algorithms or techniques that can be applied to healthcare data, which can have broader applications in other industries or fields of research.

Significance of the Study in Research Proposal

The significance of a study in a research proposal refers to the importance or relevance of the research question, problem, or objective that the study aims to address. It explains why the research is valuable, relevant, and important to the academic or scientific community, policymakers, or society at large. A strong statement of significance can help to persuade the reviewers or funders of the research proposal that the study is worth funding and conducting.

Here is an example of a significance statement in a research proposal:

Title : The Effects of Gamification on Learning Programming: A Comparative Study

Significance Statement:

This proposed study aims to investigate the effects of gamification on learning programming. With the increasing demand for computer science professionals, programming has become a fundamental skill in the computer field. However, learning programming can be challenging, and students may struggle with motivation and engagement. Gamification has emerged as a promising approach to improve students’ engagement and motivation in learning, but its effects on programming education are not yet fully understood. This study is significant because it can provide valuable insights into the potential benefits of gamification in programming education and inform the development of effective teaching strategies to enhance students’ learning outcomes and interest in programming.

Examples of Significance of the Study

Here are some examples of the significance of a study that indicates how you can write this into your research paper according to your research topic:

Research on an Improved Water Filtration System : This study has the potential to impact millions of people living in water-scarce regions or those with limited access to clean water. A more efficient and affordable water filtration system can reduce water-borne diseases and improve the overall health of communities, enabling them to lead healthier, more productive lives.

Study on the Impact of Remote Work on Employee Productivity : Given the shift towards remote work due to recent events such as the COVID-19 pandemic, this study is of considerable significance. Findings could help organizations better structure their remote work policies and offer insights on how to maximize employee productivity, wellbeing, and job satisfaction.

Investigation into the Use of Solar Power in Developing Countries : With the world increasingly moving towards renewable energy, this study could provide important data on the feasibility and benefits of implementing solar power solutions in developing countries. This could potentially stimulate economic growth, reduce reliance on non-renewable resources, and contribute to global efforts to combat climate change.

Research on New Learning Strategies in Special Education : This study has the potential to greatly impact the field of special education. By understanding the effectiveness of new learning strategies, educators can improve their curriculum to provide better support for students with learning disabilities, fostering their academic growth and social development.

Examination of Mental Health Support in the Workplace : This study could highlight the impact of mental health initiatives on employee wellbeing and productivity. It could influence organizational policies across industries, promoting the implementation of mental health programs in the workplace, ultimately leading to healthier work environments.

Evaluation of a New Cancer Treatment Method : The significance of this study could be lifesaving. The research could lead to the development of more effective cancer treatments, increasing the survival rate and quality of life for patients worldwide.

When to Write Significance of the Study

The Significance of the Study section is an integral part of a research proposal or a thesis. This section is typically written after the introduction and the literature review. In the research process, the structure typically follows this order:

• Title – The name of your research.
• Abstract – A brief summary of the entire research.
• Introduction – A presentation of the problem your research aims to solve.
• Literature Review – A review of existing research on the topic to establish what is already known and where gaps exist.
• Significance of the Study – An explanation of why the research matters and its potential impact.

In the Significance of the Study section, you will discuss why your study is important, who it benefits, and how it adds to existing knowledge or practice in your field. This section is your opportunity to convince readers, and potentially funders or supervisors, that your research is valuable and worth undertaking.

Advantages of Significance of the Study

The Significance of the Study section in a research paper has multiple advantages:

• Establishes Relevance: This section helps to articulate the importance of your research to your field of study, as well as the wider society, by explicitly stating its relevance. This makes it easier for other researchers, funders, and policymakers to understand why your work is necessary and worth supporting.
• Guides the Research: Writing the significance can help you refine your research questions and objectives. This happens as you critically think about why your research is important and how it contributes to your field.
• Attracts Funding: If you are seeking funding or support for your research, having a well-written significance of the study section can be key. It helps to convince potential funders of the value of your work.
• Opens up Further Research: By stating the significance of the study, you’re also indicating what further research could be carried out in the future, based on your work. This helps to pave the way for future studies and demonstrates that your research is a valuable addition to the field.
• Provides Practical Applications: The significance of the study section often outlines how the research can be applied in real-world situations. This can be particularly important in applied sciences, where the practical implications of research are crucial.
• Enhances Understanding: This section can help readers understand how your study fits into the broader context of your field, adding value to the existing literature and contributing new knowledge or insights.

Limitations of Significance of the Study

The Significance of the Study section plays an essential role in any research. However, it is not without potential limitations. Here are some that you should be aware of:

• Subjectivity: The importance and implications of a study can be subjective and may vary from person to person. What one researcher considers significant might be seen as less critical by others. The assessment of significance often depends on personal judgement, biases, and perspectives.
• Predictability of Impact: While you can outline the potential implications of your research in the Significance of the Study section, the actual impact can be unpredictable. Research doesn’t always yield the expected results or have the predicted impact on the field or society.
• Difficulty in Measuring: The significance of a study is often qualitative and can be challenging to measure or quantify. You can explain how you think your research will contribute to your field or society, but measuring these outcomes can be complex.
• Possibility of Overstatement: Researchers may feel pressured to amplify the potential significance of their study to attract funding or interest. This can lead to overstating the potential benefits or implications, which can harm the credibility of the study if these results are not achieved.
• Overshadowing of Limitations: Sometimes, the significance of the study may overshadow the limitations of the research. It is important to balance the potential significance with a thorough discussion of the study’s limitations.
• Dependence on Successful Implementation: The significance of the study relies on the successful implementation of the research. If the research process has flaws or unexpected issues arise, the anticipated significance might not be realized.

About the author

Muhammad Hassan

Researcher, Academic Writer, Web developer

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What is the Significance of the Study?

• By DiscoverPhDs
• August 25, 2020

• what the significance of the study means,
• why it’s important to include in your research work,
• where you would include it in your paper, thesis or dissertation,
• how you write one
• and finally an example of a well written section about the significance of the study.

What does Significance of the Study mean?

The significance of the study is a written statement that explains why your research was needed. It’s a justification of the importance of your work and impact it has on your research field, it’s contribution to new knowledge and how others will benefit from it.

Why is the Significance of the Study important?

The significance of the study, also known as the rationale of the study, is important to convey to the reader why the research work was important. This may be an academic reviewer assessing your manuscript under peer-review, an examiner reading your PhD thesis, a funder reading your grant application or another research group reading your published journal paper. Your academic writing should make clear to the reader what the significance of the research that you performed was, the contribution you made and the benefits of it.

How do you write the Significance of the Study?

When writing this section, first think about where the gaps in knowledge are in your research field. What are the areas that are poorly understood with little or no previously published literature? Or what topics have others previously published on that still require further work. This is often referred to as the problem statement.

The introduction section within the significance of the study should include you writing the problem statement and explaining to the reader where the gap in literature is.

Then think about the significance of your research and thesis study from two perspectives: (1) what is the general contribution of your research on your field and (2) what specific contribution have you made to the knowledge and who does this benefit the most.

For example, the gap in knowledge may be that the benefits of dumbbell exercises for patients recovering from a broken arm are not fully understood. You may have performed a study investigating the impact of dumbbell training in patients with fractures versus those that did not perform dumbbell exercises and shown there to be a benefit in their use. The broad significance of the study would be the improvement in the understanding of effective physiotherapy methods. Your specific contribution has been to show a significant improvement in the rate of recovery in patients with broken arms when performing certain dumbbell exercise routines.

This statement should be no more than 500 words in length when written for a thesis. Within a research paper, the statement should be shorter and around 200 words at most.

Significance of the Study: An example

Building on the above hypothetical academic study, the following is an example of a full statement of the significance of the study for you to consider when writing your own. Keep in mind though that there’s no single way of writing the perfect significance statement and it may well depend on the subject area and the study content.

Here’s another example to help demonstrate how a significance of the study can also be applied to non-technical fields:

The significance of this research lies in its potential to inform clinical practices and patient counseling. By understanding the psychological outcomes associated with non-surgical facial aesthetics, practitioners can better guide their patients in making informed decisions about their treatment plans. Additionally, this study contributes to the body of academic knowledge by providing empirical evidence on the effects of these cosmetic procedures, which have been largely anecdotal up to this point.

The statement of the significance of the study is used by students and researchers in academic writing to convey the importance of the research performed; this section is written at the end of the introduction and should describe the specific contribution made and who it benefits.

The unit of analysis refers to the main parameter that you’re investigating in your research project or study.

A thesis and dissertation appendix contains additional information which supports your main arguments. Find out what they should include and how to format them.

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What is the Significance of a Study? Examples and Guide

If you’re reading this post you’re probably wondering: what is the significance of a study?

No matter where you’re at with a piece of research, it is a good idea to think about the potential significance of your work. And sometimes you’ll have to explicitly write a statement of significance in your papers, it addition to it forming part of your thesis.

In this post I’ll cover what the significance of a study is, how to measure it, how to describe it with examples and add in some of my own experiences having now worked in research for over nine years.

If you’re reading this because you’re writing up your first paper, welcome! You may also like my how-to guide for all aspects of writing your first research paper .

Looking for guidance on writing the statement of significance for a paper or thesis? Click here to skip straight to that section.

What is the Significance of a Study?

For research papers, theses or dissertations it’s common to explicitly write a section describing the significance of the study. We’ll come onto what to include in that section in just a moment.

However the significance of a study can actually refer to several different things.

Working our way from the most technical to the broadest, depending on the context, the significance of a study may refer to:

• Within your study: Statistical significance. Can we trust the findings?
• Wider research field: Research significance. How does your study progress the field?
• Commercial / economic significance: Could there be business opportunities for your findings?
• Societal significance: What impact could your study have on the wider society.
• And probably other domain-specific significance!

We’ll shortly cover each of them in turn, including how they’re measured and some examples for each type of study significance.

But first, let’s touch on why you should consider the significance of your research at an early stage.

Why Care About the Significance of a Study?

No matter what is motivating you to carry out your research, it is sensible to think about the potential significance of your work. In the broadest sense this asks, how does the study contribute to the world?

After all, for many people research is only worth doing if it will result in some expected significance. For the vast majority of us our studies won’t be significant enough to reach the evening news, but most studies will help to enhance knowledge in a particular field and when research has at least some significance it makes for a far more fulfilling longterm pursuit.

Furthermore, a lot of us are carrying out research funded by the public. It therefore makes sense to keep an eye on what benefits the work could bring to the wider community.

Often in research you’ll come to a crossroads where you must decide which path of research to pursue. Thinking about the potential benefits of a strand of research can be useful for deciding how to spend your time, money and resources.

It’s worth noting though, that not all research activities have to work towards obvious significance. This is especially true while you’re a PhD student, where you’re figuring out what you enjoy and may simply be looking for an opportunity to learn a new skill.

However, if you’re trying to decide between two potential projects, it can be useful to weigh up the potential significance of each.

Let’s now dive into the different types of significance, starting with research significance.

Research Significance

What is the research significance of a study.

Unless someone specifies which type of significance they’re referring to, it is fair to assume that they want to know about the research significance of your study.

Research significance describes how your work has contributed to the field, how it could inform future studies and progress research.

Where should I write about my study’s significance in my thesis?

Typically you should write about your study’s significance in the Introduction and Conclusions sections of your thesis.

It’s important to mention it in the Introduction so that the relevance of your work and the potential impact and benefits it could have on the field are immediately apparent. Explaining why your work matters will help to engage readers (and examiners!) early on.

It’s also a good idea to detail the study’s significance in your Conclusions section. This adds weight to your findings and helps explain what your study contributes to the field.

On occasion you may also choose to include a brief description in your Abstract.

What is expected when submitting an article to a journal

It is common for journals to request a statement of significance, although this can sometimes be called other things such as:

• Impact statement
• Significance statement
• Advances in knowledge section

Here is one such example of what is expected:

Impact Statement:  An Impact Statement is required for all submissions.  Your impact statement will be evaluated by the Editor-in-Chief, Global Editors, and appropriate Associate Editor. For your manuscript to receive full review, the editors must be convinced that it is an important advance in for the field. The Impact Statement is not a restating of the abstract. It should address the following: Why is the work submitted important to the field? How does the work submitted advance the field? What new information does this work impart to the field? How does this new information impact the field? Experimental Biology and Medicine journal, author guidelines

Typically the impact statement will be shorter than the Abstract, around 150 words.

Defining the study’s significance is helpful not just for the impact statement (if the journal asks for one) but also for building a more compelling argument throughout your submission. For instance, usually you’ll start the Discussion section of a paper by highlighting the research significance of your work. You’ll also include a short description in your Abstract too.

How to describe the research significance of a study, with examples

Whether you’re writing a thesis or a journal article, the approach to writing about the significance of a study are broadly the same.

I’d therefore suggest using the questions above as a starting point to base your statements on.

• Why is the work submitted important to the field?
• How does the work submitted advance the field?
• What new information does this work impart to the field?
• How does this new information impact the field?

Answer those questions and you’ll have a much clearer idea of the research significance of your work.

When describing it, try to clearly state what is novel about your study’s contribution to the literature. Then go on to discuss what impact it could have on progressing the field along with recommendations for future work.

Potential sentence starters

If you’re not sure where to start, why not set a 10 minute timer and have a go at trying to finish a few of the following sentences. Not sure on what to put? Have a chat to your supervisor or lab mates and they may be able to suggest some ideas.

• This study is important to the field because…
• These findings advance the field by…
• Our results highlight the importance of…
• Our discoveries impact the field by…

Now you’ve had a go let’s have a look at some real life examples.

Statement of significance examples

A statement of significance / impact:

Impact Statement This review highlights the historical development of the concept of “ideal protein” that began in the 1950s and 1980s for poultry and swine diets, respectively, and the major conceptual deficiencies of the long-standing concept of “ideal protein” in animal nutrition based on recent advances in amino acid (AA) metabolism and functions. Nutritionists should move beyond the “ideal protein” concept to consider optimum ratios and amounts of all proteinogenic AAs in animal foods and, in the case of carnivores, also taurine. This will help formulate effective low-protein diets for livestock, poultry, and fish, while sustaining global animal production. Because they are not only species of agricultural importance, but also useful models to study the biology and diseases of humans as well as companion (e.g. dogs and cats), zoo, and extinct animals in the world, our work applies to a more general readership than the nutritionists and producers of farm animals. Wu G, Li P. The “ideal protein” concept is not ideal in animal nutrition.  Experimental Biology and Medicine . 2022;247(13):1191-1201. doi: 10.1177/15353702221082658

And the same type of section but this time called “Advances in knowledge”:

Advances in knowledge: According to the MY-RADs criteria, size measurements of focal lesions in MRI are now of relevance for response assessment in patients with monoclonal plasma cell disorders. Size changes of 1 or 2 mm are frequently observed due to uncertainty of the measurement only, while the actual focal lesion has not undergone any biological change. Size changes of at least 6 mm or more in  T 1  weighted or  T 2  weighted short tau inversion recovery sequences occur in only 5% or less of cases when the focal lesion has not undergone any biological change. Wennmann M, Grözinger M, Weru V, et al. Test-retest, inter- and intra-rater reproducibility of size measurements of focal bone marrow lesions in MRI in patients with multiple myeloma [published online ahead of print, 2023 Apr 12].  Br J Radiol . 2023;20220745. doi: 10.1259/bjr.20220745

Other examples of research significance

Moving beyond the formal statement of significance, here is how you can describe research significance more broadly within your paper.

Describing research impact in an Abstract of a paper:

Three-dimensional visualisation and quantification of the chondrocyte population within articular cartilage can be achieved across a field of view of several millimetres using laboratory-based micro-CT. The ability to map chondrocytes in 3D opens possibilities for research in fields from skeletal development through to medical device design and treatment of cartilage degeneration. Conclusions section of the abstract in my first paper .

In the Discussion section of a paper:

We report for the utility of a standard laboratory micro-CT scanner to visualise and quantify features of the chondrocyte population within intact articular cartilage in 3D. This study represents a complimentary addition to the growing body of evidence supporting the non-destructive imaging of the constituents of articular cartilage. This offers researchers the opportunity to image chondrocyte distributions in 3D without specialised synchrotron equipment, enabling investigations such as chondrocyte morphology across grades of cartilage damage, 3D strain mapping techniques such as digital volume correlation to evaluate mechanical properties  in situ , and models for 3D finite element analysis  in silico  simulations. This enables an objective quantification of chondrocyte distribution and morphology in three dimensions allowing greater insight for investigations into studies of cartilage development, degeneration and repair. One such application of our method, is as a means to provide a 3D pattern in the cartilage which, when combined with digital volume correlation, could determine 3D strain gradient measurements enabling potential treatment and repair of cartilage degeneration. Moreover, the method proposed here will allow evaluation of cartilage implanted with tissue engineered scaffolds designed to promote chondral repair, providing valuable insight into the induced regenerative process. The Discussion section of the paper is laced with references to research significance.

How is longer term research significance measured?

Looking beyond writing impact statements within papers, sometimes you’ll want to quantify the long term research significance of your work. For instance when applying for jobs.

The most obvious measure of a study’s long term research significance is the number of citations it receives from future publications. The thinking is that a study which receives more citations will have had more research impact, and therefore significance , than a study which received less citations. Citations can give a broad indication of how useful the work is to other researchers but citations aren’t really a good measure of significance.

Bear in mind that us researchers can be lazy folks and sometimes are simply looking to cite the first paper which backs up one of our claims. You can find studies which receive a lot of citations simply for packaging up the obvious in a form which can be easily found and referenced, for instance by having a catchy or optimised title.

Likewise, research activity varies wildly between fields. Therefore a certain study may have had a big impact on a particular field but receive a modest number of citations, simply because not many other researchers are working in the field.

Nevertheless, citations are a standard measure of significance and for better or worse it remains impressive for someone to be the first author of a publication receiving lots of citations.

Other measures for the research significance of a study include:

• Accolades: best paper awards at conferences, thesis awards, “most downloaded” titles for articles, press coverage.
• How much follow-on research the study creates. For instance, part of my PhD involved a novel material initially developed by another PhD student in the lab. That PhD student’s research had unlocked lots of potential new studies and now lots of people in the group were using the same material and developing it for different applications. The initial study may not receive a high number of citations yet long term it generated a lot of research activity.

That covers research significance, but you’ll often want to consider other types of significance for your study and we’ll cover those next.

Statistical Significance

What is the statistical significance of a study.

Often as part of a study you’ll carry out statistical tests and then state the statistical significance of your findings: think p-values eg <0.05. It is useful to describe the outcome of these tests within your report or paper, to give a measure of statistical significance.

Effectively you are trying to show whether the performance of your innovation is actually better than a control or baseline and not just chance. Statistical significance deserves a whole other post so I won’t go into a huge amount of depth here.

Things that make publication in  The BMJ  impossible or unlikely Internal validity/robustness of the study • It had insufficient statistical power, making interpretation difficult; • Lack of statistical power; The British Medical Journal’s guide for authors

Calculating statistical significance isn’t always necessary (or valid) for a study, such as if you have a very small number of samples, but it is a very common requirement for scientific articles.

Writing a journal article? Check the journal’s guide for authors to see what they expect. Generally if you have approximately five or more samples or replicates it makes sense to start thinking about statistical tests. Speak to your supervisor and lab mates for advice, and look at other published articles in your field.

How is statistical significance measured?

Statistical significance is quantified using p-values . Depending on your study design you’ll choose different statistical tests to compute the p-value.

A p-value of 0.05 is a common threshold value. The 0.05 means that there is a 1/20 chance that the difference in performance you’re reporting is just down to random chance.

• p-values above 0.05 mean that the result isn’t statistically significant enough to be trusted: it is too likely that the effect you’re showing is just luck.
• p-values less than or equal to 0.05 mean that the result is statistically significant. In other words: unlikely to just be chance, which is usually considered a good outcome.

Low p-values (eg p = 0.001) mean that it is highly unlikely to be random chance (1/1000 in the case of p = 0.001), therefore more statistically significant.

It is important to clarify that, although low p-values mean that your findings are statistically significant, it doesn’t automatically mean that the result is scientifically important. More on that in the next section on research significance.

How to describe the statistical significance of your study, with examples

In the first paper from my PhD I ran some statistical tests to see if different staining techniques (basically dyes) increased how well you could see cells in cow tissue using micro-CT scanning (a 3D imaging technique).

In your methods section you should mention the statistical tests you conducted and then in the results you will have statements such as:

Between mediums for the two scan protocols C/N [contrast to noise ratio] was greater for EtOH than the PBS in both scanning methods (both  p  < 0.0001) with mean differences of 1.243 (95% CI [confidence interval] 0.709 to 1.778) for absorption contrast and 6.231 (95% CI 5.772 to 6.690) for propagation contrast. … Two repeat propagation scans were taken of samples from the PTA-stained groups. No difference in mean C/N was found with either medium: PBS had a mean difference of 0.058 ( p  = 0.852, 95% CI -0.560 to 0.676), EtOH had a mean difference of 1.183 ( p  = 0.112, 95% CI 0.281 to 2.648). From the Results section of my first paper, available here . Square brackets added for this post to aid clarity.

From this text the reader can infer from the first paragraph that there was a statistically significant difference in using EtOH compared to PBS (really small p-value of <0.0001). However, from the second paragraph, the difference between two repeat scans was statistically insignificant for both PBS (p = 0.852) and EtOH (p = 0.112).

By conducting these statistical tests you have then earned your right to make bold statements, such as these from the discussion section:

Propagation phase-contrast increases the contrast of individual chondrocytes [cartilage cells] compared to using absorption contrast. From the Discussion section from the same paper.

Without statistical tests you have no evidence that your results are not just down to random chance.

Beyond describing the statistical significance of a study in the main body text of your work, you can also show it in your figures.

In figures such as bar charts you’ll often see asterisks to represent statistical significance, and “n.s.” to show differences between groups which are not statistically significant. Here is one such figure, with some subplots, from the same paper:

In this example an asterisk (*) between two bars represents p < 0.05. Two asterisks (**) represents p < 0.001 and three asterisks (***) represents p < 0.0001. This should always be stated in the caption of your figure since the values that each asterisk refers to can vary.

Now that we know if a study is showing statistically and research significance, let’s zoom out a little and consider the potential for commercial significance.

Commercial and Industrial Significance

What are commercial and industrial significance.

Moving beyond significance in relation to academia, your research may also have commercial or economic significance.

Simply put:

• Commercial significance: could the research be commercialised as a product or service? Perhaps the underlying technology described in your study could be licensed to a company or you could even start your own business using it.
• Industrial significance: more widely than just providing a product which could be sold, does your research provide insights which may affect a whole industry? Such as: revealing insights or issues with current practices, performance gains you don’t want to commercialise (e.g. solar power efficiency), providing suggested frameworks or improvements which could be employed industry-wide.

I’ve grouped these two together because there can certainly be overlap. For instance, perhaps your new technology could be commercialised whilst providing wider improvements for the whole industry.

Commercial and industrial significance are not relevant to most studies, so only write about it if you and your supervisor can think of reasonable routes to your work having an impact in these ways.

How are commercial and industrial significance measured?

Unlike statistical and research significances, the measures of commercial and industrial significance can be much more broad.

Here are some potential measures of significance:

Commercial significance:

• How much value does your technology bring to potential customers or users?
• How big is the potential market and how much revenue could the product potentially generate?
• Is the intellectual property protectable? i.e. patentable, or if not could the novelty be protected with trade secrets: if so publish your method with caution!
• If commercialised, could the product bring employment to a geographical area?

Industrial significance:

What impact could it have on the industry? For instance if you’re revealing an issue with something, such as unintended negative consequences of a drug , what does that mean for the industry and the public? This could be:

• Reduced overhead costs
• Better safety
• Faster production methods
• Improved scaleability

How to describe the commercial and industrial significance of a study, with examples

Commercial significance.

If your technology could be commercially viable, and you’ve got an interest in commercialising it yourself, it is likely that you and your university may not want to immediately publish the study in a journal.

You’ll probably want to consider routes to exploiting the technology and your university may have a “technology transfer” team to help researchers navigate the various options.

However, if instead of publishing a paper you’re submitting a thesis or dissertation then it can be useful to highlight the commercial significance of your work. In this instance you could include statements of commercial significance such as:

The measurement technology described in this study provides state of the art performance and could enable the development of low cost devices for aerospace applications. An example of commercial significance I invented for this post

Industrial significance

First, think about the industrial sectors who could benefit from the developments described in your study.

For example if you’re working to improve battery efficiency it is easy to think of how it could lead to performance gains for certain industries, like personal electronics or electric vehicles. In these instances you can describe the industrial significance relatively easily, based off your findings.

For example:

By utilising abundant materials in the described battery fabrication process we provide a framework for battery manufacturers to reduce dependence on rare earth components. Again, an invented example

For other technologies there may well be industrial applications but they are less immediately obvious and applicable. In these scenarios the best you can do is to simply reframe your research significance statement in terms of potential commercial applications in a broad way.

As a reminder: not all studies should address industrial significance, so don’t try to invent applications just for the sake of it!

Societal Significance

What is the societal significance of a study.

The most broad category of significance is the societal impact which could stem from it.

If you’re working in an applied field it may be quite easy to see a route for your research to impact society. For others, the route to societal significance may be less immediate or clear.

Studies can help with big issues facing society such as:

• Medical applications : vaccines, surgical implants, drugs, improving patient safety. For instance this medical device and drug combination I worked on which has a very direct route to societal significance.
• Political significance : Your research may provide insights which could contribute towards potential changes in policy or better understanding of issues facing society.
• Public health : for instance COVID-19 transmission and related decisions.
• Climate change : mitigation such as more efficient solar panels and lower cost battery solutions, and studying required adaptation efforts and technologies. Also, better understanding around related societal issues, for instance this study on the effects of temperature on hate speech.

How is societal significance measured?

Societal significance at a high level can be quantified by the size of its potential societal effect. Just like a lab risk assessment, you can think of it in terms of probability (or how many people it could help) and impact magnitude.

Societal impact = How many people it could help x the magnitude of the impact

Think about how widely applicable the findings are: for instance does it affect only certain people? Then think about the potential size of the impact: what kind of difference could it make to those people?

Between these two metrics you can get a pretty good overview of the potential societal significance of your research study.

How to describe the societal significance of a study, with examples

Quite often the broad societal significance of your study is what you’re setting the scene for in your Introduction. In addition to describing the existing literature, it is common to for the study’s motivation to touch on its wider impact for society.

For those of us working in healthcare research it is usually pretty easy to see a path towards societal significance.

Our CLOUT model has state-of-the-art performance in mortality prediction, surpassing other competitive NN models and a logistic regression model … Our results show that the risk factors identified by the CLOUT model agree with physicians’ assessment, suggesting that CLOUT could be used in real-world clinicalsettings. Our results strongly support that CLOUT may be a useful tool to generate clinical prediction models, especially among hospitalized and critically ill patient populations. Learning Latent Space Representations to Predict Patient Outcomes: Model Development and Validation

In other domains the societal significance may either take longer or be more indirect, meaning that it can be more difficult to describe the societal impact.

Even so, here are some examples I’ve found from studies in non-healthcare domains:

We examined food waste as an initial investigation and test of this methodology, and there is clear potential for the examination of not only other policy texts related to food waste (e.g., liability protection, tax incentives, etc.; Broad Leib et al., 2020) but related to sustainable fishing (Worm et al., 2006) and energy use (Hawken, 2017). These other areas are of obvious relevance to climate change… AI-Based Text Analysis for Evaluating Food Waste Policies

The continued development of state-of-the art NLP tools tailored to climate policy will allow climate researchers and policy makers to extract meaningful information from this growing body of text, to monitor trends over time and administrative units, and to identify potential policy improvements. BERT Classification of Paris Agreement Climate Action Plans

Top Tips For Identifying & Writing About the Significance of Your Study

• Writing a thesis? Describe the significance of your study in the Introduction and the Conclusion .
• Submitting a paper? Read the journal’s guidelines. If you’re writing a statement of significance for a journal, make sure you read any guidance they give for what they’re expecting.
• Take a step back from your research and consider your study’s main contributions.
• Read previously published studies in your field . Use this for inspiration and ideas on how to describe the significance of your own study
• Discuss the study with your supervisor and potential co-authors or collaborators and brainstorm potential types of significance for it.

Now you’ve finished reading up on the significance of a study you may also like my how-to guide for all aspects of writing your first research paper .

Writing an academic journal paper

I hope that you’ve learned something useful from this article about the significance of a study. If you have any more research-related questions let me know, I’m here to help.

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How To Write Significance of the Study (With Examples) 

Whether you’re writing a research paper or thesis, a portion called Significance of the Study ensures your readers understand the impact of your work. Learn how to effectively write this vital part of your research paper or thesis through our detailed steps, guidelines, and examples.

Related: How to Write a Concept Paper for Academic Research

Table of Contents

What is the significance of the study.

The Significance of the Study presents the importance of your research. It allows you to prove the study’s impact on your field of research, the new knowledge it contributes, and the people who will benefit from it.

Related: How To Write Scope and Delimitation of a Research Paper (With Examples)

Where Should I Put the Significance of the Study?

The Significance of the Study is part of the first chapter or the Introduction. It comes after the research’s rationale, problem statement, and hypothesis.

Related: How to Make Conceptual Framework (with Examples and Templates)

Why Should I Include the Significance of the Study?

The purpose of the Significance of the Study is to give you space to explain to your readers how exactly your research will be contributing to the literature of the field you are studying 1 . It’s where you explain why your research is worth conducting and its significance to the community, the people, and various institutions.

How To Write Significance of the Study: 5 Steps

Below are the steps and guidelines for writing your research’s Significance of the Study.

1. Use Your Research Problem as a Starting Point

Your problem statement can provide clues to your research study’s outcome and who will benefit from it 2 .

Ask yourself, “How will the answers to my research problem be beneficial?”. In this manner, you will know how valuable it is to conduct your study. 

Let’s say your research problem is “What is the level of effectiveness of the lemongrass (Cymbopogon citratus) in lowering the blood glucose level of Swiss mice (Mus musculus)?”

Discovering a positive correlation between the use of lemongrass and lower blood glucose level may lead to the following results:

• Increased public understanding of the plant’s medical properties;
• Higher appreciation of the importance of lemongrass  by the community;
• Adoption of lemongrass tea as a cheap, readily available, and natural remedy to lower their blood glucose level.

Once you’ve zeroed in on the general benefits of your study, it’s time to break it down into specific beneficiaries.

2. State How Your Research Will Contribute to the Existing Literature in the Field

Think of the things that were not explored by previous studies. Then, write how your research tackles those unexplored areas. Through this, you can convince your readers that you are studying something new and adding value to the field.

3. Explain How Your Research Will Benefit Society

In this part, tell how your research will impact society. Think of how the results of your study will change something in your community. 

For example, in the study about using lemongrass tea to lower blood glucose levels, you may indicate that through your research, the community will realize the significance of lemongrass and other herbal plants. As a result, the community will be encouraged to promote the cultivation and use of medicinal plants.

4. Mention the Specific Persons or Institutions Who Will Benefit From Your Study

Using the same example above, you may indicate that this research’s results will benefit those seeking an alternative supplement to prevent high blood glucose levels.

5. Indicate How Your Study May Help Future Studies in the Field

You must also specifically indicate how your research will be part of the literature of your field and how it will benefit future researchers. In our example above, you may indicate that through the data and analysis your research will provide, future researchers may explore other capabilities of herbal plants in preventing different diseases.

Tips and Warnings

• Think ahead . By visualizing your study in its complete form, it will be easier for you to connect the dots and identify the beneficiaries of your research.
• Write concisely. Make it straightforward, clear, and easy to understand so that the readers will appreciate the benefits of your research. Avoid making it too long and wordy.
• Go from general to specific . Like an inverted pyramid, you start from above by discussing the general contribution of your study and become more specific as you go along. For instance, if your research is about the effect of remote learning setup on the mental health of college students of a specific university , you may start by discussing the benefits of the research to society, to the educational institution, to the learning facilitators, and finally, to the students.
• Seek help . For example, you may ask your research adviser for insights on how your research may contribute to the existing literature. If you ask the right questions, your research adviser can point you in the right direction.
• Revise, revise, revise. Be ready to apply necessary changes to your research on the fly. Unexpected things require adaptability, whether it’s the respondents or variables involved in your study. There’s always room for improvement, so never assume your work is done until you have reached the finish line.

Significance of the Study Examples

This section presents examples of the Significance of the Study using the steps and guidelines presented above.

Example 1: STEM-Related Research

Research Topic: Level of Effectiveness of the Lemongrass ( Cymbopogon citratus ) Tea in Lowering the Blood Glucose Level of Swiss Mice ( Mus musculus ).

Significance of the Study .

This research will provide new insights into the medicinal benefit of lemongrass ( Cymbopogon citratus ), specifically on its hypoglycemic ability.

Through this research, the community will further realize promoting medicinal plants, especially lemongrass, as a preventive measure against various diseases. People and medical institutions may also consider lemongrass tea as an alternative supplement against hyperglycemia. 

Moreover, the analysis presented in this study will convey valuable information for future research exploring the medicinal benefits of lemongrass and other medicinal plants.  

Example 2: Business and Management-Related Research

Research Topic: A Comparative Analysis of Traditional and Social Media Marketing of Small Clothing Enterprises.

Significance of the Study:

By comparing the two marketing strategies presented by this research, there will be an expansion on the current understanding of the firms on these marketing strategies in terms of cost, acceptability, and sustainability. This study presents these marketing strategies for small clothing enterprises, giving them insights into which method is more appropriate and valuable for them. 

Specifically, this research will benefit start-up clothing enterprises in deciding which marketing strategy they should employ. Long-time clothing enterprises may also consider the result of this research to review their current marketing strategy.

Furthermore, a detailed presentation on the comparison of the marketing strategies involved in this research may serve as a tool for further studies to innovate the current method employed in the clothing Industry.

Example 3: Social Science -Related Research.

Research Topic:  Divide Et Impera : An Overview of How the Divide-and-Conquer Strategy Prevailed on Philippine Political History.

Significance of the Study :

Through the comprehensive exploration of this study on Philippine political history, the influence of the Divide et Impera, or political decentralization, on the political discernment across the history of the Philippines will be unraveled, emphasized, and scrutinized. Moreover, this research will elucidate how this principle prevailed until the current political theatre of the Philippines.

In this regard, this study will give awareness to society on how this principle might affect the current political context. Moreover, through the analysis made by this study, political entities and institutions will have a new approach to how to deal with this principle by learning about its influence in the past.

In addition, the overview presented in this research will push for new paradigms, which will be helpful for future discussion of the Divide et Impera principle and may lead to a more in-depth analysis.

Example 4: Humanities-Related Research

Research Topic: Effectiveness of Meditation on Reducing the Anxiety Levels of College Students.

Significance of the Study: 

This research will provide new perspectives in approaching anxiety issues of college students through meditation. 

Specifically, this research will benefit the following:

 Community – this study spreads awareness on recognizing anxiety as a mental health concern and how meditation can be a valuable approach to alleviating it.

Academic Institutions and Administrators – through this research, educational institutions and administrators may promote programs and advocacies regarding meditation to help students deal with their anxiety issues.

Mental health advocates – the result of this research will provide valuable information for the advocates to further their campaign on spreading awareness on dealing with various mental health issues, including anxiety, and how to stop stigmatizing those with mental health disorders.

Parents – this research may convince parents to consider programs involving meditation that may help the students deal with their anxiety issues.

Students will benefit directly from this research as its findings may encourage them to consider meditation to lower anxiety levels.

Future researchers – this study covers information involving meditation as an approach to reducing anxiety levels. Thus, the result of this study can be used for future discussions on the capabilities of meditation in alleviating other mental health concerns.

Frequently Asked Questions

1. what is the difference between the significance of the study and the rationale of the study.

Both aim to justify the conduct of the research. However, the Significance of the Study focuses on the specific benefits of your research in the field, society, and various people and institutions. On the other hand, the Rationale of the Study gives context on why the researcher initiated the conduct of the study.

Let’s take the research about the Effectiveness of Meditation in Reducing Anxiety Levels of College Students as an example. Suppose you are writing about the Significance of the Study. In that case, you must explain how your research will help society, the academic institution, and students deal with anxiety issues through meditation. Meanwhile, for the Rationale of the Study, you may state that due to the prevalence of anxiety attacks among college students, you’ve decided to make it the focal point of your research work.

2. What is the difference between Justification and the Significance of the Study?

In Justification, you express the logical reasoning behind the conduct of the study. On the other hand, the Significance of the Study aims to present to your readers the specific benefits your research will contribute to the field you are studying, community, people, and institutions.

Suppose again that your research is about the Effectiveness of Meditation in Reducing the Anxiety Levels of College Students. Suppose you are writing the Significance of the Study. In that case, you may state that your research will provide new insights and evidence regarding meditation’s ability to reduce college students’ anxiety levels. Meanwhile, you may note in the Justification that studies are saying how people used meditation in dealing with their mental health concerns. You may also indicate how meditation is a feasible approach to managing anxiety using the analysis presented by previous literature.

3. How should I start my research’s Significance of the Study section?

– This research will contribute… – The findings of this research… – This study aims to… – This study will provide… – Through the analysis presented in this study… – This study will benefit…

Moreover, you may start the Significance of the Study by elaborating on the contribution of your research in the field you are studying.

4. What is the difference between the Purpose of the Study and the Significance of the Study?

The Purpose of the Study focuses on why your research was conducted, while the Significance of the Study tells how the results of your research will benefit anyone.

Suppose your research is about the Effectiveness of Lemongrass Tea in Lowering the Blood Glucose Level of Swiss Mice . You may include in your Significance of the Study that the research results will provide new information and analysis on the medical ability of lemongrass to solve hyperglycemia. Meanwhile, you may include in your Purpose of the Study that your research wants to provide a cheaper and natural way to lower blood glucose levels since commercial supplements are expensive.

5. What is the Significance of the Study in Tagalog?

In Filipino research, the Significance of the Study is referred to as Kahalagahan ng Pag-aaral.

• Draft your Significance of the Study. Retrieved 18 April 2021, from http://dissertationedd.usc.edu/draft-your-significance-of-the-study.html
• Regoniel, P. (2015). Two Tips on How to Write the Significance of the Study. Retrieved 18 April 2021, from https://simplyeducate.me/2015/02/09/significance-of-the-study/

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Jewel Kyle Fabula

Jewel Kyle Fabula is a Bachelor of Science in Economics student at the University of the Philippines Diliman. His passion for learning mathematics developed as he competed in some mathematics competitions during his Junior High School years. He loves cats, playing video games, and listening to music.

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Establishing Rationale and Significance of Research

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This chapter builds on the first five chapters in this handbook that explained the research design typology. The focus here is on establishing rationale and significance of research. This chapter is intended to serve as a guide for practitioners to apply and integrate the research design typology layers into a scholarly manuscript. In contrast to the broad scope of the first five chapters, this chapter concentrates on how to integrate specific components of the typology regardless of which ideology the researcher holds on the continuum (positivist, post-positivist, pragmatist, interpretivist, or constructivist).

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Research Questions and Research Design

Reflections on Methodological Issues

Katz, M. (2010). Toward a new moral paradigm in health care delivery: Accounting for individuals. American Journal Of Law and Medicine , 36 (1), 78–135.

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Matteson, L. & Lacey F. M. (2011). Doing your literature review: Traditional and systematic techniques . London: Sage

Mclean, T. R. (2006). The future of telemedicine and its Faustian reliance on regulatory trade barriers for protection. Health Matrix: Journal of Law-Medicine , 16 (2), 443–509.

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The Ethics of Using QI Methods to Improve Health Quality and Safety (2006). Hastings Center Report , Vol. 36, pp. S1–S40.

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Hahn, J. (2015). Establishing Rationale and Significance of Research. In: Strang, K.D. (eds) The Palgrave Handbook of Research Design in Business and Management. Palgrave Macmillan, New York. https://doi.org/10.1057/9781137484956_7

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• Published: 14 August 2024

Nonlinear dynamics of multi-omics profiles during human aging

• Xiaotao Shen   ORCID: orcid.org/0000-0002-9608-9964 1 , 2 , 3   na1 ,
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• Wenyu Zhou 1 ,
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• Si Wu 1 &
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• Systems biology

Aging is a complex process associated with nearly all diseases. Understanding the molecular changes underlying aging and identifying therapeutic targets for aging-related diseases are crucial for increasing healthspan. Although many studies have explored linear changes during aging, the prevalence of aging-related diseases and mortality risk accelerates after specific time points, indicating the importance of studying nonlinear molecular changes. In this study, we performed comprehensive multi-omics profiling on a longitudinal human cohort of 108 participants, aged between 25 years and 75 years. The participants resided in California, United States, and were tracked for a median period of 1.7 years, with a maximum follow-up duration of 6.8 years. The analysis revealed consistent nonlinear patterns in molecular markers of aging, with substantial dysregulation occurring at two major periods occurring at approximately 44 years and 60 years of chronological age. Distinct molecules and functional pathways associated with these periods were also identified, such as immune regulation and carbohydrate metabolism that shifted during the 60-year transition and cardiovascular disease, lipid and alcohol metabolism changes at the 40-year transition. Overall, this research demonstrates that functions and risks of aging-related diseases change nonlinearly across the human lifespan and provides insights into the molecular and biological pathways involved in these changes.

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Personal aging markers and ageotypes revealed by deep longitudinal profiling

Considerations for reproducible omics in aging research

Principal component-based clinical aging clocks identify signatures of healthy aging and targets for clinical intervention

Aging is a complex and multifactorial process of physiological changes strongly associated with various human diseases, including cardiovascular diseases (CVDs), diabetes, neurodegeneration and cancer 1 . The alterations of molecules (including transcripts, proteins, metabolites and cytokines) are critically important to understand the underlying mechanism of aging and discover potential therapeutic targets for aging-related diseases. Recently, the development of high-throughput omics technologies has enabled researchers to study molecular changes at the system level 2 . A growing number of studies have comprehensively explored the molecular changes that occur during aging using omics profiling 3 , 4 , and most focus on linear changes 5 . It is widely recognized that the occurrence of aging-related diseases does not follow a proportional increase with age. Instead, the risk of these diseases accelerates at specific points throughout the human lifespan 6 . For example, in the United States, the prevalence of CVDs (encompassing atherosclerosis, stroke and myocardial infarction) is approximately 40% between the ages of 40 and 59, increases to about 75% between 60 and 79 and reaches approximately 86% in individuals older than 80 years 7 . Similarly, also in the United States, the prevalence of neurodegenerative diseases, such as Parkinson’s disease and Alzheimer’s disease, exhibits an upward trend as well as human aging progresses, with distinct turning points occurring around the ages of 40 and 65, respectively 8 , 9 , 10 . Some studies also found that brain aging followed an accelerated decline in flies 11 and chimpanzees 12 that lived past middle age and advanced age.

The observation of a nonlinear increase in the prevalence of aging-related diseases implies that the process of human aging is not a simple linear trend. Consequently, investigating the nonlinear changes in molecules will likely reveal previously unreported molecular signatures and mechanistic insights. Some studies examined the nonlinear alterations of molecules during human aging 13 . For instance, nonlinear changes in RNA and protein expression related to aging have been documented 14 , 15 , 16 . Moreover, certain DNA methylation sites have exhibited nonlinear changes in methylation intensity during aging, following a power law pattern 17 . Li et al. 18 identified the 30s and 50s as transitional periods during women’s aging. Although aging patterns are thought to reflect the underlying biological mechanisms, the comprehensive landscape of nonlinear changes of different types of molecules during aging remains largely unexplored. Remarkably, the global monitoring of nonlinear changing molecular profiles throughout human aging has yet to be fully used to extract basic insights into the biology of aging.

In the present study, we conducted a comprehensive deep multi-omics profiling on a longitudinal human cohort comprising 108 individuals aged from 25 years to 75 years. The cohort was followed over a span of several years (median, 1.7 years), with the longest monitoring period for a single participant reaching 6.8 years (2,471 days). Various types of omics data were collected from the participants’ biological samples, including transcriptomics, proteomics, metabolomics, cytokines, clinical laboratory tests, lipidomics, stool microbiome, skin microbiome, oral microbiome and nasal microbiome. The investigation explored the changes occurring across different omics profiles during human aging. Remarkably, many molecular markers and biological pathways exhibited a nonlinear pattern throughout the aging process, thereby providing valuable insight into periods of dramatic alterations during human aging.

Most of the molecules change nonlinearly during aging

We collected longitudinal biological samples from 108 participants over several years, with a median tracking period of 1.7 years and a maximum period of 6.8 years, and conducted multi-omics profiling on the samples. The participants were sampled every 3–6 months while healthy and had diverse ethnic backgrounds and ages ranging from 25 years to 75 years (median, 55.7 years). The participants’ body mass index (BMI) ranged from 19.1 kg m −2 to 40.8 kg m −2 (median, 28.2 kg m −2 ). Among the participants, 51.9% were female (Fig. 1a and Extended Data Fig. 1a–d ). For each visit, we collected blood, stool, skin swab, oral swab and nasal swab samples. In total, 5,405 biological samples (including 1,440 blood samples, 926 stool samples, 1,116 skin swab samples, 1,001 oral swab samples and 922 nasal swab samples) were collected. The biological samples were used for multi-omics data acquisition (including transcriptomics from peripheral blood mononuclear cells (PBMCs), proteomics from plasma, metabolomics from plasma, cytokines from plasma, clinical laboratory tests from plasma, lipidomics from plasma, stool microbiome, skin microbiome, oral microbiome and nasal microbiome; Methods ). In total, 135,239 biological features (including 10,346 transcripts, 302 proteins, 814 metabolites, 66 cytokines, 51 clinical laboratory tests, 846 lipids, 52,460 gut microbiome taxons, 8,947 skin microbiome taxons, 8,947 oral microbiome taxons and 52,460 nasal microbiome taxons) were acquired, resulting in 246,507,456,400 data points (Fig. 1b and Extended Data Fig. 1e,f ). The average sampling period and number of samples for each participant were 626 days and 47 samples, respectively. Notably, one participant was deeply monitored for 6.8 years (2,471 days), during which 367 samples were collected (Fig. 1c ). Overall, this extensive and longitudinal multi-omics dataset enables us to examine the molecular changes that occur during the human aging process. The detailed characteristics of all participants are provided in the Supplementary Data . For each participant, the omics data were aggregated and averaged across all healthy samples to represent the individual’s mean value, as detailed in the Methods section. Compared to cross-sectional cohorts, which have only a one-time point sample from each participant, our longitudinal dataset, which includes multiple time point samples from each participant, is more robust for detecting complex aging-related changes in molecules and functions. This is because analysis of multi-time point samples can detect participants’ baseline and robustly evaluate individuals’ longitudinal molecular changes.

a , The demographics of the 108 participants in the study are presented. b , Sample collection and multi-omics data acquisition of the cohort. Four types of biological samples were collected, and 10 types of omics data were acquired. c , Collection time range and sample numbers for each participant. The top x axis represents the collection range for each participant (read line), and the bottom x axis represents the sample number for each participant (bar plot). Bars are color-coded by omics type. d , Significantly changed molecules and microbes during aging were detected using the Spearman correlation approach ( P  < 0.05). The P values were not adjusted ( Methods ). Dots are color-coded by omics type. e , Differential expressional molecules/microbes in different age ranges compared to baseline (25–40 years old, two-sided Wilcoxon test, P  < 0.05). The P values were not adjusted ( Methods ). f , The linear changing molecules comprised only a small part of dysregulated molecules in at least one age range. g , Heatmap depicting the nonlinear changing molecules and microbes during human aging.

We included samples only from healthy visits and adjusted for confounding factors (for example, BMI, sex, insulin resistance/insulin sensitivity (IRIS) and ethnicity; Extended Data Fig. 1a–d ), allowing us to discern the molecules and microbes genuinely associated with aging ( Methods ). Two common and traditional approaches, linear regression and Spearman correlation, were first used to identify the linear changing molecules during human aging 5 . The linear regression method is commonly used for linear changing molecules. As expected, both approaches have very high consistent results for each type of omics data (Supplementary Fig. 1a ). For convenience, the Spearman correlation approach was used in the analysis. Interestingly, only a small portion of all the molecules and microbes (749 out of 11,305, 6.6%; only genus level was used for microbiome data; Methods ) linearly changed during human aging (Fig. 1d and Supplementary Fig. 1b ), consistent with our previous studies 5 ( Methods ). Next, we examined nonlinear effects by categorizing all participants into distinct age stages according to their ages and investigated the dysregulated molecules within each age stage compared to the baseline (25–40 years old; Methods ). Interestingly, using this approach, 81.03% of molecules (9,106 out of 11,305) exhibited changes in at least one age stage compared to the baseline (Fig. 1e and Extended Data Fig. 2a ). Remarkably, the percentage of linear changing molecules was relatively small compared to the overall dysregulated molecules during aging (mean, 16.2%) (Fig. 1f and Extended Data Fig. 2b ). To corroborate our findings, we employed a permutation approach to calculate permutated P values, which yielded consistent results ( Methods ). The heatmap depicting all dysregulated molecules also clearly illustrates pronounced nonlinear changes (Fig. 1g ). Taken together, these findings strongly suggest that a substantial number of molecules and microbes undergo nonlinear changes throughout human aging.

Clustering reveals nonlinear multi-omics changes during aging

Next, we assessed whether the multi-omics data collected from the longitudinal cohort could serve as reliable indicators of the aging process. Our analysis revealed a substantial correlation between a significant proportion of the omics data and the ages of the participants (Fig. 2a ). Particularly noteworthy was the observation that, among all the omics data examined, metabolomics, cytokine and oral microbiome data displayed the strongest association with age (Fig. 2a and Extended Data Fig. 3a–c ). Partial least squares (PLS) regression was further used to compare the strength of the age effect across different omics data types. The results are consistent with the results presented above in Fig. 2a ( Methods ). These findings suggest the potential utility of these datasets as indicators of the aging process while acknowledging that further research is needed for validation 4 . As the omics data are not accurately matched across all the samples, we then smoothed the omics data using our previously published approach 19 ( Methods and Supplementary Fig. 2a–c ). Next, to reveal the specific patterns of molecules that change during human aging, we then grouped all the molecules with similar trajectories using an unsupervised fuzzy c-means clustering approach 19 ( Methods , Fig. 3b and Supplementary Fig. 2d,e ). We identified 11 clusters of molecular trajectories that changed during aging, which ranged in size from 638 to 1,580 molecules/microbes (Supplementary Fig. 2f and Supplementary Data ). We found that most molecular patterns exhibit nonlinear changes, indicating that aging is not a linear process (Fig. 2b ). Among the 11 identified clusters, three distinct clusters (2, 4 and 5) displayed compelling, straightforward and easily understandable patterns that spanned the entire lifespan (Fig. 2c ). Most molecules within these three clusters primarily consist of transcripts (Supplementary Fig. 2f ), which is expected because transcripts dominate the multi-omics data (8,556 out of 11,305, 75.7%). Cluster 4 exhibits a relatively stable pattern until approximately 60 years of age, after which it shows a rapid decrease (Fig. 2c ). Conversely, clusters 2 and 5 display fluctuations before 60 years of age, followed by a sharp increase and an upper inflection point at approximately 55–60 years of age (Fig. 2c ). We also attempted to observe this pattern of molecular change during aging individually. The participant with the longest follow-up period of 6.8 years (Fig. 1c ) approached the age of 60 years (range, 59.5–66.3 years; Extended Data Fig. 1g ), and it was not possible to identify obvious patterns in this short time window (Supplementary Fig. 2g ). Tracking individuals longitudinally over longer periods (decades) will be required to observe these trajectories at an individual level.

a , Spearman correlation (cor) between the first principal component and ages for each type of omics data. The shaded area around the regression line represents the 95% confidence interval. b , The heatmap shows the molecular trajectories in 11 clusters during human aging. The right stacked bar plots show the percentages of different kinds of omics data, and the right box plots show the correlation distribution between features and ages ( n  = 108 participants). c , Three notable clusters of molecules that exhibit clear and straightforward nonlinear changes during human aging. The top stacked bar plots show the percentages of different kinds of omics data, and the top box plots show the correlation distribution between features and ages ( n  = 108 participants). The box plot shows the median (line), interquartile range (IQR) (box) and whiskers extending to 1.5 × IQR. Bars and lines are color-coded by omics type. Abs, absolute.

a , Pathway enrichment and module analysis for each transcriptome cluster. The left panel is the heatmap for the pathways that undergo nonlinear changes across aging. The right panel is the pathway similarity network ( Methods ) ( n  = 108 participants). b , Pathway enrichment for metabolomics in each cluster. Enriched pathways and related metabolites are illustrated (Benjamini–Hochberg-adjusted P  < 0.05). c , Four clinical laboratory tests that change during human aging: blood urea nitrogen, serum/plasma glucose, mean corpuscular hemoglobin and red cell distribution width ( n  = 108 participants). The box plot shows the median (line), interquartile range (IQR) (box) and whiskers extending to 1.5 × IQR.

Although confounders, including sex, were corrected before analysis ( Methods ), we acknowledge that the age range for menopause in females is typically between 45 years and 55 years of age 20 , which is very close to the major transition points in all three clusters (Fig. 2c ). Therefore, we conducted further investigation into whether the menopausal status of females in the dataset contributed to the observed transition point at approximately 55 years of age (Fig. 2c ) by performing separate clustering analyses on the male and female datasets. Surprisingly, both the male and female datasets exhibited similar clusters, as illustrated in Extended Data Fig. 4a . This suggests that the transition point observed at approximately 55 years of age is not solely attributed to female menopause but, rather, represents a common phenomenon in the aging process of both sexes. This result is consistent with previous studies 14 , 15 , further supporting the notion that this transition point is a major characteristic feature of human aging. Moreover, to investigate the possibility that the transcriptomics data might skew the results toward transcriptomic changes as age-related factors, we conducted two additional clustering analyses—one focusing solely on transcriptomic data and another excluding it. Interestingly, both analyses yielded nearly identical three-cluster configurations, as observed using the complete omics dataset (Extended Data Fig. 4b ). This reinforces the robustness of the identified clusters and confirms that they are consistent across various omics platforms, not just driven by transcriptomic data.

Nonlinear changes in function and disease risk during aging

To gain further insight into the biological functions associated with the nonlinear changing molecules within the three identified clusters, we conducted separate functional analyses for transcriptomics, proteomics and metabolomics datasets for all three clusters. In brief, we constructed a similarity network using enriched pathways from various databases (Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome) and identified modules to eliminate redundant annotations. We then used all modules from different databases to reduce redundancy further using the same approach and define the final functional modules ( Methods , Extended Data Fig. 4c and Supplementary Data ). We identified some functional modules that were reported in previous studies, but we defined their more accurate patterns of change during human aging. Additionally, we also found previously unreported potential functional modules during human aging ( Supplementary Data ). For instance, in cluster 2, we identified a transcriptomic module associated with GTPase activity (adjusted P  = 1.64 × 10 −6 ) and histone modification (adjusted P   =  6.36 × 10 −7 ) (Fig. 3a ). Because we lack epigenomic data in this study, our findings should be validated through additional experiments in the future. GTPase activity is closely correlated with programmed cell death (apoptosis), and some previous studies showed that this activity increases during aging 21 . Additionally, histone modifications have been demonstrated to increase during human aging 22 . In cluster 4, we identified one transcriptomics module associated with oxidative stress; this module includes antioxidant activity, oxygen carrier activity, oxygen binding and peroxidase activity (adjusted P  = 0.029) (Fig. 3a ). Previous studies demonstrated that oxidative stress and many reactive oxygen species (ROS) are positively associated with increased inflammation in relation to aging 23 . In cluster 5, the first transcriptomics module is associated with mRNA stability, which includes mRNA destabilization (adjusted P   =  0.0032), mRNA processing (adjusted P   =  3.2 × 10 −4 ), positive regulation of the mRNA catabolic process (adjusted P   =  1.46 × 10 −4 ) and positive regulation of the mRNA metabolic process (adjusted P   =  0.00177) (Fig. 3a ). Previous studies showed that mRNA turnover is associated with aging 24 . The second module is associated with autophagy (Fig. 3a ), which increases during human aging, as demonstrated in previous studies 25 .

In addition, we also identified certain modules in the clusters that suggest a nonlinear increase in several disease risks during human aging. For instance, in cluster 2, where components increase gradually and then rapidly after age 60, the phenylalanine metabolism pathway (adjusted P   =  4.95 × 10 −4 ) was identified (Fig. 3b ). Previous studies showed that aging is associated with a progressive increase in plasma phenylalanine levels concomitant with cardiac dysfunction, and dysregulated phenylalanine catabolism is a factor that triggers deviations from healthy cardiac aging trajectories 26 . Additionally, C-X-C motif chemokine 5 (CXCL5 or ENA78) from proteomics data, which has higher concentrations in atherosclerosis 27 , is also detected in cluster 2 ( Supplementary Data ). The clinical laboratory test blood urea nitrogen, which provides important information about kidney function, is also detected in cluster 2 (Fig. 3c ). This indicates that kidney function nonlinearly decreases during aging. Furthermore, the clinical laboratory test for serum/plasma glucose, a marker of type 2 diabetes (T2D), falls within cluster 2. This is consistent with and supported by many previous studies demonstrating that aging is a major risk factor for T2D 28 . Collectively, these findings suggest a nonlinear escalation in the risk of cardiovascular and kidney diseases and T2D with advancing age, particularly after the age of 60 years (Fig. 2c ).

The identified modules in cluster 4 also indicate a nonlinear increase in disease risks. For instance, the unsaturated fatty acids biosynthesis pathway (adjusted P   =  4.71 × 10 −7 ) is decreased in cluster 4. Studies have shown that unsaturated fatty acids are helpful in reducing CVD risk and maintaining brain function 29 , 30 . The pathway of alpha-linolenic acid and linolenic acid metabolism (adjusted P   =  1.32 × 10 −4 ) can reduce aging-associated diseases, such as CVD 31 . We also detected the caffeine metabolism pathway (adjusted P   =  7.34 × 10 −5 ) in cluster 4, which suggests that the ability to metabolize caffeine decreases during aging. Additionally, the cytokine MCP1 (chemokine (C-C motif) ligand 2 (CCL2)), a member of the CC chemokine family, plays an important immune regulatory role and is also in cluster 4 ( Supplementary Data ). These findings further support previous observations and highlight the nonlinear increase in age-related disease risk as individuals age.

Cluster 5 comprises the clinical tests of mean corpuscular hemoglobin and red cell distribution width (Fig. 3c ). These tests assess the average hemoglobin content per red blood cell and the variability in the size and volume of red blood cells, respectively. These findings align with the aforementioned transcriptomic data, which suggest a nonlinear reduction in the oxygen-carrying capacity associated with the aging process.

Aside from these three distinct clusters (Fig. 2c ), we also conducted pathway enrichment analysis across all other eight clusters, which displayed highly nonlinear trajectories, employing the same method (Fig. 2b and Supplementary Data ). Notably, cluster 11 exhibited a consistent increase up until the age of 50, followed by a decline until the age of 56, after which no substantial changes were observed up to the age of 75. A particular transcriptomics module related to DNA repair was identified, encompassing three pathways: positive regulation of double-strand break repair (adjusted P   =  0.042), peptidyl−lysine acetylation (adjusted P   =  1.36 × 10 −5 ) and histone acetylation (adjusted P   =  3.45 × 10 −4 ) (Extended Data Fig. 4d ). These three pathways are critical in genomic stability, gene expression and metabolic balances, with their levels diminishing across the human lifespan 32 , 33 , 34 . Our findings reveal a nonlinear alteration across the human lifespan in these pathways, indicating an enhancement in DNA repair capabilities before the age of 50, a marked reduction between the ages of 50 and 56 and stabilization after that until the age of 75. The pathway enrichment results for all clusters are detailed in the Supplementary Data .

Altogether, the comprehensive functional analysis offers valuable insights into the nonlinear changes observed in molecular profiles and their correlations with biological functions and disease risks across the human lifespan. Our findings reveal that individuals aged 60 and older exhibit increased susceptibility to CVD, kidney issues and T2D. These results carry important implications for both the diagnosis and prevention of these diseases. Notably, many clinically actionable markers were identified, which have the potential for improved healthcare management and enhanced overall well-being of the aging population.

Uncovering waves of aging-related molecules during aging

Although the trajectory clustering approaches described above effectively identify nonlinear changing molecules and microbes that exhibit clear and compelling patterns throughout human aging, it may not be as effective in capturing substantial changes that occur at specific chronological aging periods. In such cases, alternative analytical approaches may be necessary to detect and characterize these dynamics. To gain a comprehensive understanding of changes in multi-omics profiling during human aging, we used a modified version of the DE-SWAN algorithm 14 , as described in the Methods section. This algorithm identifies dysregulated molecules and microbes throughout the human lifespan by analyzing molecule levels within 20-year windows and comparing two groups in 10-year parcels while sliding the window incrementally from young to old ages 14 . Using this approach and multiomics data, we detected changes at specific stages of lifespan and uncovered the sequential effects of aging. Our analysis revealed thousands of molecules exhibiting changing patterns throughout aging, forming distinct waves, as illustrated in Fig. 3a . Notably, we observed two prominent crests occurring around the ages of 45 and 65, respectively (Fig. 4a ). Notably, too, these crests were consistent with findings from a previous study that included only proteomics data 14 . Specifically, crest 2 aligns with our previous trajectory clustering result, indicating a turning point at approximately 60 years of age (Fig. 2c ).

a , Number of molecules and microbes differentially expressed during aging. Two local crests at the ages of 44 years and 60 years were identified. b , c , The same waves were detected using different q value ( b ) and window ( c ) cutoffs. d , The number of molecules/microbes differentially expressed for different types of omics data during human aging.

To demonstrate the significance of the two crests, we employed different q value cutoffs and sliding window parameters, which consistently revealed the same detectable waves (Fig. 4b,c and Supplementary Fig. 4a,b ). Furthermore, when we permuted the ages of individuals, the crests disappeared (Supplementary Figs. 3a and 4c ) ( Methods ). These observations highlight the robustness of the two major waves of aging-related molecular changes across the human lifespan. Although we already accounted for confounders before our statistical analysis, we took additional steps to explore their impact. Specifically, we investigated whether confounders, such as insulin sensitivity, sex and ethnicity, differed between the two crests across various age ranges. As anticipated, these confounders did not show significant differences across other age brackets (Supplementary Fig. 4d ). This further supports our conclusion that the observed differences in the two crests are attributable to age rather than other confounding variables.

The identified crests represent notable milestones in the aging process and suggest specific age ranges where substantial molecular alterations occur. Therefore, we investigated the age-related waves for each type of omics data. Interestingly, most types of omics data exhibited two distinct crests that were highly robust (Fig. 3b and Supplementary Fig. 4 ). Notably, the proteomics data displayed two age-related crests at ages around 40 years and 60 years. Only a small overlap was observed between our dataset and the results from the previous study (1,305 proteins versus 302 proteins, with only 75 proteins overlapping). The observed pattern in our study was largely consistent with the previous findings 14 . However, our finding that many types of omics data, including transcriptomics, proteomics, metabolomics, cytokine, gut microbiome, skin microbiome and nasal microbiome, exhibit these waves, often with a similar pattern as the proteomics data (Fig. 4d ), supports the hypothesis that aging-related changes are not limited to a specific omics layer but, rather, involve a coordinated and systemic alteration across multiple molecular components. Identifying consistent crests across different omics data underscores the robustness and reliability of these molecular milestones in the aging process.

Next, we investigated the roles and functions of dysregulated molecules within two distinct crests. Notably, we found that the two crests related to aging predominantly consisted of the same molecules (Supplementary Fig. 6 ). To focus on the unique biological functions associated with each crest and eliminate commonly occurring molecules, we removed background molecules present in most stages. To explore the specific biological functions associated with each type of omics data (transcriptomics, proteomics and metabolomics) for both crests, we employed the function annotation approach described above ( Methods ). In brief, we constructed a similarity network of enriched pathways and identified modules to remove redundant annotations (Supplementary Fig. 6 and Extended Data Fig. 5a,b ). Furthermore, we applied the same approach to all modules to reduce redundancy and identify the final functional modules ( Methods and Extended Data Fig. 6a ). Our analysis revealed significant changes in multiple modules associated with the two crests (Extended Data Fig. 6b–d ). To present the results clearly, Fig. 5a displays the top 20 pathways (according to adjusted P value) for each type of omics data, and the Supplementary Data provides a comprehensive list of all identified functional modules.

a , Pathway enrichment and biological functional module analysis for crests 1 and 2. Dots and lines are color-coded by omics type. b , The overlapping of molecules between two crests and three clusters.

Interestingly, the analysis identifies many dysregulated functional modules in crests 1 and 2, indicating a nonlinear risk for aging-related diseases. In particular, several modules associated with CVD were identified in both crest 1 and crest 2 (Fig. 5a ), which is consistent with the above results (Fig. 3b ). For instance, the dysregulation of platelet degranulation (crest 1: adjusted P   =  1.77 × 10 −30 ; crest 2: adjusted P   =  1.73 × 10 −26 ) 35 , 36 , complement cascade (crest 1: adjusted P   =  3.84 × 10 −30 ; crest 2: adjusted P   =  2.02 × 10 −28 ), complement and coagulation cascades (crest 1: adjusted P   =  1.78 × 10 −46 ; crest 2: adjusted P   =  2.02 × 10 −28 ) 37 , 38 , protein activation cascade (crest 1: adjusted P   =  1.56 × 10 −17 ; crest 2: adjusted P   =  1.61 × 10 −8 ) and protease binding (crest 1: adjusted P   =  2.7 × 10 −6 ; crest 2: adjusted P   =  0.0114) 39 have various effects on the cardiovascular system and can contribute to various CVDs. Furthermore, blood coagulation (crest 1: adjusted P   =  1.48 × 10 −28 ; crest 2: adjusted P   =  9.10 × 10 −17 ) and fibrinolysis (crest 1: adjusted P   =  2.11 × 10 −15 ; crest 2: adjusted P   =  1.64 × 10 −10 ) were also identified, which are essential processes for maintaining blood fluidity, and dysregulation in these modules can lead to thrombotic and cardiovascular events 40 , 41 . We also identified certain dysregulated metabolic modules associated with CVD. For example, aging has been linked to an incremental rise in plasma phenylalanine levels (crest 1: adjusted P   =  9.214 × 10 −4 ; crest 2: adjusted P   =  0.0453), which can contribute to the development of cardiac hypertrophy, fibrosis and dysfunction 26 . Branched-chain amino acids (BCAAs), including valine, leucine and isoleucine (crest 1: adjusted P : not significant (NS); crest 2: adjusted P   =  0.0173), have also been implicated in CVD development 42 , 43 and T2D, highlighting their relevance in CVD pathophysiology. Furthermore, research suggests that alpha-linolenic acid (ALA) and linoleic acid metabolism (crest 1: adjusted P : NS; crest 2: adjusted P   =  0.0217) may be protective against coronary heart disease 44 , 45 . Our investigation also identified lipid metabolism modules that are associated with CVD, including high-density lipoprotein (HDL) remodeling (crest 1: adjusted P   =  1.073 × 10 −8 ; crest 2: adjusted P   =  2.589 × 10 −9 ) and glycerophospholipid metabolism (crest 1: adjusted P : NS; crest 2: adjusted P   =  0.0033), which influence various CVDs 46 , 47 , 48 .

In addition, the dysregulation of skin and muscle stability was found to be increased at crest 1 and crest 2, as evidenced by the identification of numerous modules associated with these processes (Fig. 5a,b ). This suggests that the aging of skin and muscle is markedly accelerated at crest 1 and crest 2. The extracellular matrix (ECM) provides structural stability, mechanical strength, elasticity and hydration to the tissues and cells, and the ECM of the skin is mainly composed of collagen, elastin and glycosaminoglycans (GAGs) 49 . Phosphatidylinositols are a class of phospholipids that have various roles in cytoskeleton organization 50 . Notably, the dysregulation of ECM structural constituent (crest 1: adjusted P   =  3.32 × 10 −8 ; crest 2: adjusted P   =  1.61 × 10 −8 ), GAG binding (crest 1: adjusted P   =  1.805 × 10 −8 ; crest 2: adjusted P   =  4.093 × 10 −6 ) and phosphatidylinositol binding (crest 1: adjusted P   =  3.391 × 10 −6 ; crest 2: adjusted P   =  7.832 × 10 −6 ) were identified 51 , 52 . We also identified cytolysis (crest 1: adjusted P   =  2.973 × 10 −5 ; crest 2: adjusted P : NS), which can increase water loss 53 . The dysregulated actin binding (crest 1: adjusted P   =  3.536 × 10 −8 ; crest 2: adjusted P   =  3.435 × 10 −9 ), actin filament organization (crest 1: adjusted P   =  8.406 × 10 −9 ; crest 2: adjusted P   =  1.157 × 10 −9 ) and regulation of actin cytoskeleton (crest 1: adjusted P   =  0.00090242; crest 2: adjusted P   =  6.788 × 10 −4 ) were identified, which affect the structure and function of various tissues 54 , 55 , 56 , 57 , 58 . Additionally, cell adhesion is the attachment of a cell to another cell or to ECM via adhesion molecules 59 . We identified the positive regulation of cell adhesion (crest 1: adjusted P   =  3.618 × 10 −5 ; crest 2: adjusted P   =  8.272 × 10 −9 ) module, which can prevent or delay skin aging 60 , 61 . Threonine can affect sialic acid production, which is involved in cell adhesion 62 . We also identified the glycine, serine and threonine metabolism (crest 1: adjusted P : NS; crest 2: adjusted P   =  0.00506) 62 . Additionally, scavenging of heme from plasma was identified (crest 1: adjusted P   =  1.176 × 10 −11 ; crest 2: adjusted P   =  1.694 × 10 −8 ), which can modulate skin aging as excess-free heme can damage cellular components 63 , 64 . Rho GTPases regulate a wide range of cellular responses, including changes to the cytoskeleton and cell adhesion (RHO GTPase cycle, crest 1: adjusted P   =  9.956 × 10 −10 ; crest 2: adjusted P   =  1.546 × 10 −5 ) 65 . In relation to muscle, previous studies demonstrated that muscle mass decreases by approximately 3–8% per decade after the age of 30, with an even higher decline rate after the age of 60, which consistently coincides with the observed second crest 66 . Interestingly, we identified dysregulation in the module associated with the structural constituent of muscle (crest 1: adjusted P   =  0.00565; crest 2: adjusted P   =  0.0162), consistent with previous findings 66 . Furthermore, we identified the pathway associated with caffeine metabolism (crest 1: adjusted P   =  0.00378; crest 2: adjusted P   =  0.0162), which is consistent with our observations above (Fig. 2b ) and implies that the capacity to metabolize caffeine undergoes a notable alteration not only around 60 years of age but also around the age of 40 years.

In crest 1, we identified specific modules associated with lipid and alcohol metabolism. Previous studies established that lipid metabolism declines with human aging 67 . Our analysis revealed several modules related to lipid metabolism, including plasma lipoprotein remodeling (crest 1: adjusted P   =  2.269 × 10 −9 ), chylomicron assembly (crest 1: adjusted P   =  9.065 × 10 −7 ) and ATP-binding cassette (ABC) transporters (adjusted P   =  1.102 × 10 −4 ). Moreover, we discovered a module linked to alcohol metabolism (alcohol binding, adjusted P   =  8.485 × 10 −7 ), suggesting a decline in alcohol metabolization efficiency with advancing age, particularly around the age of 40, when it significantly diminishes. In crest 2, we observed prominent modules related to immune dysfunction, encompassing acute-phase response (adjusted P   =  2.851 × 10 −8 ), antimicrobial humoral response (adjusted P   =  2.181 × 10 −5 ), zymogen activation (adjusted P   =  4.367 × 10 −6 ), complement binding (adjusted P   =  0.002568), mononuclear cell differentiation (adjusted P   =  9.352 × 10 −8 ), viral process (adjusted P   =  5.124 × 10 −7 ) and regulation of hemopoiesis (adjusted P   =  3.522 × 10 −7 ) (Fig. 5a ). Age-related changes in the immune system, collectively known as immunosenescence, have been extensively documented 68 , 69 , 70 , and our results demonstrate a rapid decline at age 60. Furthermore, we also identified modules associated with kidney function (glomerular filtration, adjusted P   =  0.00869) and carbohydrate metabolism (carbohydrate binding, adjusted P   =  0.01045). Our previous findings indicated a decline in kidney function around the age of 60 years (Fig. 3c ), as did the present result of this observation. Previous studies indicated the influence of carbohydrates on aging, characterized by the progressive decline of physiological functions and increased susceptibility to diseases over time 71 , 72 .

In summary, our analysis identifies many dysregulated functional modules identified in both crest 1 and crest 2 that underlie the risk for various diseases and alterations of biological functions. Notably, we observed an overlap of dysregulated functional modules among clusters 2, 4 and 6 because they overlap at the molecular level, as depicted in Fig. 5b . This indicates that certain molecular components are shared among these clusters and the identified crests. However, it is important to note that numerous molecules are specific to each of the two approaches employed in our study. This suggests that these two approaches complement each other in identifying nonlinear changes in molecules and functions during human aging. By using both approaches, we were able to capture a more comprehensive understanding of the molecular alterations associated with aging and their potential implications for diseases.

Analyzing a longitudinal multi-omics dataset involving 108 participants, we successfully captured the dynamic and nonlinear molecular changes that occur during human aging. Our study’s strength lies in the comprehensive nature of the dataset, which includes multiple time point samples for each participant. This longitudinal design enhances the reliability and robustness of our findings compared to cross-sectional studies with only one time point sample for each participant. The first particularly intriguing finding from our analysis is that only a small fraction of molecules (6.6%) displayed linear changes throughout human aging (Fig. 1d ). This observation is consistent with previous research and underscores the limitations of relying solely on linear regression to understand the complexity of aging-related molecular changes 5 . Instead, our study revealed that a considerable number of molecules (81.0%) exhibited nonlinear patterns (Fig. 1e ). Notably, this nonlinear trend was observed across all types of omics data with remarkably high consistency (Fig. 1e,g ), highlighting the widespread functionally relevant nature of these dynamic changes. By unveiling the nonlinear molecular alterations associated with aging, our research contributes to a more comprehensive understanding of the aging process and its molecular underpinnings.

To further investigate the nonlinear changing molecules observed in our study, we employed a trajectory clustering approach to group molecules with similar temporal patterns. This analysis revealed the presence of three distinct clusters (Fig. 2c ) that exhibited clear and compelling patterns across the human lifespan. These clusters suggest that there are specific age ranges, such as around 60 years old, where distinct and extensive molecular changes occur (Fig. 2c ). Functional analysis revealed several modules that exhibited nonlinear changes during human aging. For example, we identified a module associated with oxidative stress, which is consistent with previous studies linking oxidative stress to the aging process 23 (Fig. 3a ). Our analysis indicates that this pathway increases significantly after the age of 60 years. In cluster 5, we identified a transcriptomics module associated with mRNA stabilization and autophagy (Fig. 3a ). Both of these processes have been implicated in the aging process and are involved in maintaining cellular homeostasis and removing damaged components. Furthermore, our analysis uncovered nonlinear changes in disease risk across aging. In cluster 2, we identified the phenylalanine metabolism pathway (Fig. 3b ), which has been associated with cardiac dysfunction during aging 26 . Additionally, we found that the clinical laboratory tests blood urea nitrogen and serum/plasma glucose increase significantly with age (cluster 2; Fig. 3c ), indicating a nonlinear decline in kidney function and an increased risk of T2D with age, with a critical threshold occurring approximately at the age of 60 years. In cluster 4, we identified pathways related to cardiovascular health, such as the biosynthesis of unsaturated fatty acids and caffeine metabolism (Fig. 3b ). Overall, our study provides compelling evidence for the existence of nonlinear changes in molecular profiles during human aging. By elucidating the specific functional modules and disease-related pathways that exhibit such nonlinear changes, we contribute to a better understanding of the complex molecular dynamics underlying the aging process and its implications for disease risk.

Although the trajectory clustering approach proves effective in identifying molecules that undergo nonlinear changes, it may not be as proficient in capturing substantial alterations that occur at specific time points without exhibiting a consistent pattern in other stages. We then employed a modified version of the DE-SWAN algorithm 14 to comprehensively investigate changes in multi-omics profiling throughout human aging. This approach enabled us to identify waves of dysregulated molecules and microbes across the human lifespan. Our analysis revealed two prominent crests occurring around the ages of 40 years and 60 years, which were consistent across various omics data types, suggesting their universal nature (Fig. 4a,e ). Notably, in the proteomics data, we observed crests around the ages of 40 years and 60 years, which aligns approximately with a previous study (which reported crests at ages 34 years, 60 years and 78 years) 14 . Due to the age range of our cohort being 25–75 years, we did not detect the third peak. Furthermore, the differences in proteomics data acquisition platforms (mass spectrometry versus SomaScan) 14 , 73 resulted in different identified proteins, with only a small overlap (1,305 proteins versus 302 proteins, of which only 75 were shared). This discrepancy may explain the age variation of the first crest identified in the two studies (approximately 10 years). However, despite the differences in the two proteomics datasets, the wave patterns observed in both studies were highly similar 14 (Fig. 4a ). Remarkably, by considering multiple omics data types, we consistently identified similar crests for each type, indicating the universality of these waves of change across plasma molecules and microbes from various body sites (Fig. 4e and Supplementary Fig. 3 ).

The analysis of molecular functionality in the two distinct crests revealed the presence of several modules, indicating a nonlinear increase in the risks of various diseases (Fig. 5a ). Both crest 1 and crest 2 exhibit the identification of multiple modules associated with CVD, which aligns with the aforementioned findings (Fig. 3b ). Moreover, we observed an escalated dysregulation in skin and muscle functioning in both crest 1 and crest 2. Additionally, we identified a pathway linked to caffeine metabolism, indicating a noticeable alteration in caffeine metabolization not only around the age of 60 but also around the age of 40. This shift may be due to either a metabolic shift or a change in caffeine consumption. In crest 1, we also identified specific modules associated with lipid and alcohol metabolism, whereas crest 2 demonstrated prominent modules related to immune dysfunction. Furthermore, we also detected modules associated with kidney function and carbohydrate metabolism, which is consistent with our above results. These findings reinforce our previous observations regarding a decline in kidney function around the age of 60 years (Fig. 3c ) while shedding light on the impact of dysregulated functional modules in both crest 1 and crest 2, suggesting nonlinear changes in disease risk and functional dysregulation. Notably, we identified an overlap of dysregulated functional modules among clusters 2, 4 and 6, indicating molecular-level similarities between these clusters and the identified crests (Fig. 5b ). This suggests the presence of shared molecular components among these clusters and crests. However, it is crucial to note that there are also numerous molecules specific to each of the two approaches employed in our study, indicating that these approaches complement each other in identifying nonlinear changes in molecules and functions during human aging.

The present research is subject to certain constraints. We accounted for many basic characteristics (confounders) of participants in the cohort; but because this study primarily reflects between-individual differences, there may be additional confounders due to the different age distributions of the participants. For example, we identified a notable decrease in oxygen carrier activity around age 60 (Figs. 2c and 3a ) and marked variations in alcohol and caffeine metabolism around ages 40 and 60 (Fig. 3a ). However, these findings might be shaped by participants’ lifestyle—that is, physical activity and their alcohol and caffeine intake. Regrettably, we do not have such detailed behavioral data for the entire group, necessitating validation in upcoming research. Although initial BMI and insulin sensitivity measurements were available at cohort entry, subsequent metrics during the observation span were absent, marking a study limitation.

A further constraint is our cohort’s modest size, encompassing merely 108 individuals (eight individuals between 25 years and 40 years of age), which hampers the full utilization of deep learning and may affect the robustness of the identification of nonlinear changing features in Fig. 1e . Although advanced computational techniques, including deep learning, are pivotal for probing nonlinear patterns, our sample size poses restrictions. Expanding the cohort size in subsequent research would be instrumental in harnessing the full potential of machine learning tools. Another limitation of our study is that the recruitment of participants was within the community around Stanford University, driven by rigorous sample collection procedures and the substantial expenses associated with setting up a longitudinal cohort. Although our participants exhibited a considerable degree of ethnic age and biological sex diversity (Fig. 1a and Supplementary Data ), it is important to acknowledge that our cohort may not fully represent the diversity of the broader population. The selectivity of our cohort limits the generalizability of our findings. Future studies should aim to include a more diverse cohort to enhance the external validity and applicability of the results.

In addition, the mean observation span for participants was 626 days, which is insufficient for detailed inflection point analyses. Our cohort’s age range of 25–70 years lacks individuals who lie outside of this range. The molecular nonlinearity detected might be subject to inherent variations or oscillations, a factor to consider during interpretation. Our analysis has not delved into the nuances of the dynamical systems theory, which provides a robust mathematical framework for understanding observed behaviors. Delving into this theory in future endeavors may yield enhanced clarity and interpretation of the data.

Moreover, it should be noted that, in our study, the observed nonlinear molecular changes occurred across individuals of varying ages rather than within the same individuals. This is attributed to the fact that, despite our longitudinal study, the follow-up period for our participants was relatively brief for following aging patterns (median, 1.7 years; Extended Data Fig. 1g ). Such a timeframe is inadequate for detecting nonlinear molecular changes that unfold over decades throughout the human lifespan. Addressing this limitation in future research is essential.

Lastly, our study’s molecular data are derived exclusively from blood samples, casting doubt on its direct relevance to specific tissues, such as the skin or muscles. We propose that blood gene expression variations might hint at overarching physiological alterations, potentially impacting the ECM in tissues, including skin and muscle. Notably, some blood-based biomarkers and transcripts have demonstrated correlations with tissue modifications, inflammation and other elements influencing the ECM across diverse tissues 74 , 75 .

In our future endeavors, the definitive confirmation of our findings hinges on determining if nonlinear molecular patterns align with nonlinear changes in functional capacities, disease occurrences and mortality hazards. For a holistic grasp of this, amalgamating multifaceted data from long-term cohort studies covering several decades becomes crucial. Such data should encompass molecular markers, comprehensive medical records, functional assessments and mortality data. Moreover, employing cutting-edge statistical techniques is vital to intricately decipher the ties between these nonlinear molecular paths and health-centric results.

In summary, the unique contribution of our study lies not merely in reaffirming the nonlinear nature of aging but also in the depth and breadth of the multi-omics data that we analyzed. Our study goes beyond stating that aging is nonlinear by identifying specific patterns, inflection points and potential waves in aging across multiple layers of biological data during human aging. Identifying specific clusters with distinct patterns, functional implications and disease risks enhances our understanding of the aging process. By considering the nonlinear dynamics of aging-related changes, we can gain insights into specific periods of significant changes (around age 40 and age 60) and the molecular mechanisms underlying age-related diseases, which could lead to the development of early diagnosis and prevention strategies. These comprehensive multi-omics data and the approach allow for a more nuanced understanding of the complexities involved in the aging process, which we think adds value to the existing body of research. However, further research is needed to validate and expand upon these findings, potentially incorporating larger cohorts to capture the full complexity of aging.

The participant recruitment, sample collection, data acquisition and data processing were documented in previous studies conducted by Zhou et al. 76 , Ahadi et al. 5 , Schüssler-Fiorenza Rose et al. 77 , Hornburg et al. 78 and Zhou et al. 79 .

Participant recruitment

Participants provided informed written consent for the study under research protocol 23602, which was approved by the Stanford University institutional review board. This study adheres to all relevant ethical regulations, ensuring informed consents were obtained from all participants. All participants consented to publication of potentially identifiable information. The cohort comprised 108 participants who underwent follow-up assessments. Exclusion criteria encompassed conditions such as anemia, kidney disease, a history of CVD, cancer, chronic inflammation or psychiatric illnesses as well as any prior bariatric surgery or liposuction. Each participant who met the eligibility criteria and provided informed consent underwent a one-time modified insulin suppression test to quantify insulin-mediated glucose uptake at the beginning of the enrollment 76 . The steady-state plasma glucose (SSPG) levels served as a direct indicator of each individual’s insulin sensitivity in processing a glucose load. We categorized individuals with SSPG levels below 150 mg dl −1 as insulin sensitive and those with levels of 150 mg dl −1 or higher as insulin resistant 80 , 81 . Thirty-eight participants were missing SSPG values, rendering their insulin resistance or sensitivity status undetermined. We also collected fasting plasma glucose (FPG) data for 69 participants at enrollment. Based on the FPG levels, two participants were identified as having diabetes at enrollment, with FPG levels exceeding 126 mg dl −1 ( Supplementary Data ). Additionally, we measured hemoglobin A1C (HbA1C) levels during each visit, using it as a marker for average glucose levels over the past 3 months: 6.5% or higher indicates diabetes. Accordingly, four participants developed diabetes during the study period. At the beginning of the enrollment, BMI was also measured for each participant. Participants received no compensation.

Comprehensive sample collection was conducted during the follow-up period, and multi-omics data were acquired (Fig. 1b ). For each visit, the participants self-reported as healthy or non-healthy 76 . To ensure accuracy and minimize the impact of confounding factors, only samples from individuals classified as healthy were selected for subsequent analysis.

Transcriptomics

Transcriptomic profiling was conducted on flash-frozen PBMCs. RNA isolation was performed using a QIAGEN All Prep kit. Subsequently, RNA libraries were assembled using an input of 500 ng of total RNA. In brief, ribosomal RNA (rRNA) was selectively eliminated from the total RNA pool, followed by purification and fragmentation. Reverse transcription was carried out using a random primer outfitted with an Illumina-specific adaptor to yield a cDNA library. A terminal tagging procedure was used to incorporate a second adaptor sequence. The final cDNA library underwent amplification. RNA sequencing libraries underwent sequencing on an Illumina HiSeq 2000 platform. Library quantification was performed via an Agilent Bioanalyzer and Qubit fluorometric quantification (Thermo Fisher Scientific) using a high-sensitivity dsDNA kit. After normalization, barcoded libraries were pooled at equimolar ratios into a multiplexed sequencing library. An average of 5–6 libraries were processed per HiSeq 2000 lane. Standard Illumina pipelines were employed for image analysis and base calling. Read alignment to the hg19 reference genome and personal exomes was achieved using the TopHat package, followed by transcript assembly and expression quantification via HTseq and DESeq2. In the realm of data pre-processing, genes with an average read count across all samples lower than 0.5 were excluded. Samples exhibiting an average read count lower than 0.5 across all remaining genes were likewise removed. For subsequent global variance and correlation assessments, genes with an average read count of less than 1 were eliminated.

Plasma sample tryptic peptides were fractionated using a NanoLC 425 System (SCIEX) operating at a flow rate of 5 μl min −1 under a trap-elute configuration with a 0.5 × 10 mm ChromXP column (SCIEX). The liquid chromatography gradient was programmed for a 43-min run, transitioning from 4% to 32% of mobile phase B, with an overall run time of 1 h. Mobile phase A consisted of water with 0.1% formic acid, and mobile phase B was formulated with 100% acetonitrile and 0.1% formic acid. An 8-μg aliquot of non-depleted plasma was loaded onto a 15-cm ChromXP column. Mass spectrometry analysis was executed employing SWATH acquisition on a TripleTOF 6600 system. A set of 100 variable Q1 window SWATH acquisition methods was designed in high-sensitivity tandem mass spectrometry (MS/MS) mode. Subsequent data analysis included statistical scoring of peak groups from individual runs via pyProphet 82 , followed by multi-run alignment through TRIC60, ultimately generating a finalized data matrix with a false discovery rate (FDR) of 1% at the peptide level and 10% at the protein level. Protein quantitation was based on the sum of the three most abundant peptide signals for each protein. Batch effect normalization was achieved by subtracting principal components that primarily exhibited batch-associated variation, using Perseus software v.1.4.2.40.

Untargeted metabolomics

A ternary solvent system of acetone, acetonitrile and methanol in a 1:1:1 ratio was used for metabolite extraction. The extracted metabolites were dried under a nitrogen atmosphere and reconstituted in a 1:1 methanol:water mixture before analysis. Metabolite profiles were generated using both hydrophilic interaction chromatography (HILIC) and reverse-phase liquid chromatography (RPLC) under positive and negative ion modes. Thermo Q Exactive Plus mass spectrometers were employed for HILIC and RPLC analyses, respectively, in full MS scan mode. MS/MS data were acquired using quality control (QC) samples. For the HILIC separations, a ZIC-HILIC column was used with mobile phase solutions of 10 mM ammonium acetate in 50:50 and 95:5 acetonitrile:water ratios. In the case of RPLC, a Zorbax SBaq column was used, and the mobile phase consisted of 0.06% acetic acid in water and methanol. Metabolic feature detection was performed using Progenesis QI software. Features from blanks and those lacking sufficient linearity upon dilution were excluded. Only features appearing in more than 33% of the samples were retained for subsequent analyses, and any missing values were imputed using the k -nearest neighbors approach. We employed locally estimated scatterplot smoothing (LOESS) normalization 83 to correct the metabolite-specific signal drift over time. The metid package 84 was used for metabolite annotation.

Cytokine data

A panel of 62 human cytokines, chemokines and growth factors was analyzed in EDTA-anticoagulated plasma samples using Luminex-based multiplex assays with conjugated antibodies (Affymetrix). Raw fluorescence measurements were standardized to median fluorescence intensity values and subsequently subjected to variance-stabilizing transformation to account for batch-related variations. As previously reported 76 , data points characterized by background noise, termed CHEX, that deviate beyond five standard deviations from the mean (mean ± 5 × s.d.) were excluded from the analyses.

Clinical laboratory test

The tests encompassed a comprehensive metabolic panel, a full blood count, glucose and HbA1C levels, insulin assays, high-sensitivity C-reactive protein (hsCRP), immunoglobulin M (IgM) and lipid, kidney and liver panels.

Lipid extraction and quantification procedures were executed in accordance with established protocols 78 . In summary, complex lipids were isolated from 40 μl of EDTA plasma using a solvent mixture comprising methyl tertiary-butyl ether, methanol and water, followed by a biphasic separation. Subsequent lipid analysis was conducted on the Lipidyzer platform, incorporating a differential mobility spectrometry device (SelexION Technology) and a QTRAP 5500 mass spectrometer (SCIEX).

Immediately after arrival, samples were stored at −80 °C. Stool and nasal samples were processed and sequenced in-house at the Jackson Laboratory for Genomic Medicine, whereas oral and skin samples were outsourced to uBiome for additional processing. Skin and oral samples underwent 30 min of beads-beating lysis, followed by a silica-guanidinium thiocyanate-based nucleic acid isolation protocol. The V4 region of the 16S rRNA gene was amplified using specific primers, after which the DNA was barcoded and sequenced on an Illumina NextSeq 500 platform via a 2 × 150-bp paired-end protocol. Similarly, stool and nasal samples were processed for 16S rRNA V1–V3 region amplification using a different set of primers and sequenced on an Illumina MiSeq platform. For data processing, the raw sequencing data were demultiplexed using BCL2FASTQ software and subsequently filtered for quality. Reads with a Q-score lower than 30 were excluded. The DADA2 R package was used for further sequence data processing, which included filtering out reads with ambiguous bases and errors, removing chimeras and aligning sequences against a validated 16S rRNA gene database. Relative abundance calculations for amplicon sequence variants (ASVs) were performed, and samples with inadequate sequencing depth (<1,000 reads) were excluded. Local outlier factor (LOF) was calculated for each point on a depth-richness plot, and samples with abnormal LOF were removed. In summary, rigorous procedures were followed in both the collection and processing stages, leveraging automated systems and specialized software to ensure the quality and integrity of the microbiome data across multiple body sites.

Statistics and reproducibility

For all data processing, statistical analysis and data visualization tasks, RStudio, along with R language (v.4.2.1), was employed. A comprehensive list of the packages used can be found in the Supplementary Note . The Benjamini–Hochberg method was employed to account for multiple comparisons. Spearman correlation coefficients were calculated using the R functions ‘cor’ and ‘cor.test’. Principal-component analysis (PCA) was conducted using the R function ‘princomp’. Before all the analyses, the confounders, such as BMI, sex, IRIS and ethnicity, were adjusted using the previously published method 19 . In brief, we used the intensity of each feature as the dependent variable (Y) and the confounding factors as the independent variables (X) to build a linear regression model. The residuals from this model were then used as the adjusted values for that specific feature.

All the omics data were acquired randomly. No statistical methods were used to predetermine the sample size, but our sample sizes are similar to those reported in previous publications 5 , 76 , 77 , 78 , 79 , and no data were excluded from the analyses. Additionally, the investigators were blinded to allocation during experiments and outcome assessment to the conditions of the experiments. Data distribution was assumed to be normal, but this was not formally tested.

The icons used in figures are from iconfont.cn, which can be used for non-commercial purposes under the MIT license ( https://pub.dev/packages/iconfont/license ).

Cross-sectional dataset generation

The ‘cross-sectional’ dataset was created by briefly extracting information from the longitudinal dataset. The mean value was calculated to represent each molecule’s intensity for each participant. Similarly, the age of each participant was determined by calculating the mean value of ages across all sample collection time points.

Linear changing molecule detection

We detected linear changing molecules during human aging using Spearman correlation and linear regression modeling. The confounders, such as BMI, sex, IRIS and ethnicity, were adjusted using the previously published method 19 . Our analysis revealed a high correlation between these two approaches in identifying such molecules. Based on these findings, we used the Spearman correlation approach to showcase the linear changing molecules during human aging. The permutation test was also used to get the permutated P values for each feature. In brief, each feature was subjected to sample label shuffling followed by a recalculation of the Spearman correlation. This process was reiterated 10,000 times, yielding 10,000 permuted Spearman correlations. The original Spearman correlation was then compared against these permuted values to obtain the permuted P values.

Dysregulated molecules compared to baseline during human aging

To depict the dysregulated molecules during human aging compared to the baseline, we categorized the participants into different age stages based on their ages. The baseline stage was defined as individuals aged 25–40 years. For each age stage group, we employed the Wilcoxon test to identify dysregulated molecules in comparison to the baseline, considering a significance threshold of P  < 0.05. Before the statistical analysis, all the confounders were corrected. Subsequently, we visualized the resulting dysregulated molecules at different age stages using a Sankey plot. The permutation test was also used to get the permutated P values for each feature. In brief, we shuffled the sample labels and recalculated the absolute mean difference between the two groups, against which the actual absolute mean difference was benchmarked to derive the permuted P values. To identify the molecules and microbes that exhibited significant changes at any given age stage, we adjusted the P values for each feature by multiplying them by 6. This adjustment adheres to the Bonferroni correction method, ensuring a rigorous evaluation of statistical significance.

Evaluation of the age reflected by different types of omics data

To assess whether each type of omics data accurately reflects the ages of individuals in our dataset, we conducted a PCA. Subsequently, we computed the Spearman correlation coefficient between the ages of participants and the first principal component (PC1). The absolute value of this coefficient was used to evaluate the degree to which the omics data reflect the ages (Fig. 2a ). PLS regression was also used to compare the strength of the age effect to the different omics data types. In brief, the ‘pls’ function from the R package mixOmics was used to construct the regression model between omics data and ages. Then, the ‘perf’ function was used to assess the performance of all the modules with sevenfold cross-validation. The R 2 was extracted to assess the strength of the age effect on the different omics data types.

To accommodate the varying time points of biological and omics data, we employed the LOESS approach. This approach allowed us to smooth and predict the multi-omics data at specific time points (that is, every half year) 14 , 85 . In brief, for each molecule, we fitted a LOESS regression model. During the fitting process, the LOESS argument ‘span’ was optimized through cross-validation. This ensured that the LOESS model provided an accurate and non-overfitting fit to the data (Supplementary Fig. 2a,b ). Once we obtained the LOESS prediction model, we applied it to predict the intensity of each molecule at every half-year time point.

Trajectory clustering analysis

To conduct trajectory clustering analysis, we employed the fuzzy c-means clustering approach available in the R package ‘Mfuzz.’ This approach was previously described in our publication 19 . The analysis proceeded in several steps. First, the omics data were auto-scaled to ensure comparable ranges. Next, we computed the minimum centroid distances for a range of cluster numbers, specifically from 2 to 22, in step 1. These minimum centroid distances served as a cluster validity index, helping us determine the optimal cluster number. Based on predefined rules, we selected the optimal cluster number. To refine the accuracy of this selection, we merged clusters with center expression data correlations greater than 0.8 into a single cluster. This step aimed to capture similar patterns within the data. The resulting optimal cluster number was then used for the fuzzy c-means clustering. Only molecules with memberships above 0.5 were retained within each cluster for further analysis. This threshold ensured that the molecules exhibited a strong association with their assigned cluster and contributed considerably to the cluster’s characteristics.

Pathway enrichment analysis and functional module identification

Transcriptomics and proteomics pathway enrichment.

Pathway enrichment analysis was conducted using the ‘clusterProfiler’ R package 86 . The GO, KEGG and Reactome databases were used. The P values were adjusted using the Benjamini–Hochberg method, with a significance threshold set at <0.05. To minimize redundant enriched pathways and GO terms, we employed a series of analyses. First, for enriched GO terms, we used the ‘Wang’ algorithm from the R package ‘simplifyEnrichment’ to calculate the similarity between GO terms. Only connections with a similarity score greater than 0.7 were retained to construct the GO term similarity network. Subsequently, community analysis was performed using the ‘igraph’ R package to partition the network into distinct modules. The GO term with the smallest enrichment adjusted P value was chosen as the representative within each module. The same approach was applied to the enriched KEGG and Reactome pathways, with one slight modification. In this case, the ‘jaccard’ algorithm was used to calculate the similarity between pathways, and a similarity cutoff of 0.5 was employed for the Jaccard index. After removing redundant enriched pathways, we combined all the remaining GO terms and pathways. Subsequently, we calculated the similarity between these merged entities using the Jaccard index. This similarity analysis aimed to capture the overlap and relationships between the different GO terms and pathways. Using the same approach as before, we performed community analysis to identify distinct biological functional modules based on the merged GO terms and pathways.

Identification of functional modules

First, we used the ‘Wang’ algorithm for the GO database and the ‘jaccard’ algorithm for the KEGG and Reactome databases to calculate the similarity between pathways. The enriched pathways served as nodes in a similarity network, with edges representing the similarity between two nodes. Next, we employed the R package ‘igraph’ to identify modules within the network based on edge betweenness. By gradually removing edges with the highest edge betweenness scores, we constructed a hierarchical map known as a dendrogram, representing a rooted tree of the graph. The leaf nodes correspond to individual pathways, and the root node represents the entire graph 87 . We then merged pathways within each module, selecting the pathway with the smallest adjusted P value to represent the module. After this step, we merged pathways from all three databases into modules. Subsequently, we repeated the process by calculating the similarity between modules from all three databases using the ‘jaccard’ algorithm. Once again, we employed the same approach described above to identify the functional modules.

Metabolomics pathway enrichment

To perform pathway enrichment analysis for metabolomics data, we used the human KEGG pathway database. This database was obtained from KEGG using the R package ‘massDatabase’ 88 . For pathway enrichment analysis, we employed the hypergeometric distribution test from the ‘TidyMass’ project 89 . This statistical test allowed us to assess the enrichment of metabolites within each pathway. To account for multiple tests, P values were adjusted using the Benjamini–Hochberg method. We considered pathways with Benjamini–Hochberg-adjusted P values lower than 0.05 as significantly enriched.

Modified DE-SWAN

The DE-SWAN algorithm 14 was used. To begin, a unique age is selected as the center of a 20-year window. Molecule levels in individuals younger than and older than that age are compared using the Wilcoxon test to assess differential expression. P values are calculated for each molecule, indicating the significance of the observed differences. To ensure sufficient sample sizes for statistical analysis in each time window, the initial window ranges from ages 25 to 50. The left half of this window covers ages 25–40, whereas the right half spans ages 41–50. The window then moves in one-year steps; this is why Fig. 4 displays an age range of 40–65 years. To account for multiple comparisons, these P values are adjusted using Benjamini–Hochberg correction. To evaluate the robustness and relevance of the DE-SWAN results, the algorithm is tested with various parcel widths, including 15 years, 20 years, 25 years and 30 years. Additionally, different q value thresholds, such as <0.0001, <0.001, <0.01 and <0.05, are applied. By comparing the results obtained with these different parameters to results obtained by chance, we can assess the significance of the findings. To generate random results for comparison, the phenotypes of the individuals are randomly permuted, and the modified DE-SWAN algorithm is applied to the permuted dataset. This allows us to determine whether the observed results obtained with DE-SWAN are statistically significant and not merely a result of chance.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The raw data used in this study can be accessed without any restrictions on the National Institutes of Health Human Microbiome 2 project site ( https://portal.hmpdacc.org ). Both the raw and processed data are also available on the Stanford iPOP site ( http://med.stanford.edu/ipop.html ). Researchers and interested individuals can visit these websites to access the data. For further details and inquiries about the study, we recommend contacting the corresponding author, who can provide additional information and address any specific questions related to the research.

Code availability

The statistical analysis and data processing in this study were performed using R v.4.2.1, along with various base packages and additional packages. Detailed information about the specific packages used can be found in the Supplementary Note , which accompanies the manuscript. Furthermore, all the custom scripts developed for this study have been made openly accessible and can be found on the GitHub repository at https://github.com/jaspershen-lab/ipop_aging . By visiting this repository, researchers and interested individuals can access and use the custom scripts for their own analyses or to replicate the study’s findings.

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Acknowledgements

We sincerely thank all the research participants for their dedicated involvement in this study. We also thank A. Chen and L. Stainton for their valuable administrative assistance. Additionally, we are deeply grateful to A.T. Brunger’s support for this work. This work was supported by National Institutes of Health (NIH) grants U54DK102556 (M.P.S.), R01 DK110186-03 (M.P.S.), R01HG008164 (M.P.S.), NIH S10OD020141 (M.P.S.), UL1 TR001085 (M.P.S.) and P30DK116074 (M.P.S.) and by the Stanford Data Science Initiative (M.P.S.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

These authors contributed equally: Xiaotao Shen, Chuchu Wang.

Authors and Affiliations

Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA

Xiaotao Shen, Xin Zhou, Wenyu Zhou, Daniel Hornburg, Si Wu & Michael P. Snyder

Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore

Xiaotao Shen

School of Chemistry, Chemical Engineering and Biotechnology, Singapore, Singapore

Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA

Chuchu Wang

Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA

Stanford Center for Genomics and Personalized Medicine, Stanford, CA, USA

Xin Zhou & Michael P. Snyder

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Contributions

X.S. and M.P.S. conceptualized and designed the study. X.Z. and W.Z. prepared the microbiome data. D.H. and W.S. prepared the lipidomics data. X.S. and C.W. conducted the data analysis. X.S. and C.W. prepared the figures. X.S., C.W. and M.P.S. contributed to the writing and revision of the manuscript, with input from other authors. M.S. and X.S. supervised the overall study.

Corresponding author

Correspondence to Michael P. Snyder .

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Competing interests.

M.P.S. is a co-founder of Personalis, SensOmics, Qbio, January AI, Filtricine, Protos and NiMo and is on the scientific advisory boards of Personalis, SensOmics, Qbio, January AI, Filtricine, Protos, NiMo and Genapsys. D.H. has a financial interest in PrognomIQ and Seer. All other authors have no competing interests.

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Nature Aging thanks Daniel Belsky and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended data fig. 1 demographic data of all the participants in the study..

a , The ages positively correlate with BMI. The shaded area around the regression line represents the 95% confidence interval. b , Gender with age. c , Ethnicity with age. d , Insulin response with age. e , biological sample collection for all the participants. f , Overlap of the different kinds of omics data. g , The age range for each participant in this study.

Extended Data Fig. 2 Most of the molecules change nonlinearly during human aging.

a , Differential expressional microbes in different age ranges compared to baselines (25 – 40 years old, two-sided Wilcoxon test, p -value < 0.05). b , Most of the linear changing molecules and microbiota are also included in the molecules/microbes that significantly dysregulated at least one age range.

Extended Data Fig. 3 Omics data can represent aging.

PCA score plot of metabolomics data ( a ), cytokine ( b ), and oral microbiome ( c ).

Extended Data Fig. 4 Functional analysis of molecules in different clusters.

a , The Jaccard index between clusters from different datasets. b , The overlap between clusters using different types of omics data. c , Functional module detection and identification. d , Functional analysis of nonlinear changing molecules for all clusters.

Extended Data Fig. 5 Function annotation for significantly dysregulated molecules in crest 1 and 2.

a , Transcriptomics data. b , Proteomics data. c , Metabolomics data.

Extended Data Fig. 6 Pathways enrichment results for crest 1 and 2.

a , The final functional modules identified for Crest 1 and 2. b , The pathway enrichment analysis results for transcriptomics data. c , The pathway enrichment analysis results for proteomics data. d , The pathway enrichment results for metabolomics data.

Supplementary information

Supplementary figs. 1–6, reporting summary, supplementary data analysis results of the study., rights and permissions.

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Shen, X., Wang, C., Zhou, X. et al. Nonlinear dynamics of multi-omics profiles during human aging. Nat Aging (2024). https://doi.org/10.1038/s43587-024-00692-2

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Received : 09 December 2023

Accepted : 22 July 2024

Published : 14 August 2024

DOI : https://doi.org/10.1038/s43587-024-00692-2

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