RESEARCH ARTICLE
Dose response of running on blood
biomarkers of wellness in generally healthy
individuals
Bartek NogalID1☯, Svetlana Vinogradova1☯, Milena Jorge1, Ali Torkamani2,3, Paul Fabian1,
Gil Blander1*
1 InsideTracker, Cambridge, Massachusetts, United States of America, 2 The Scripps Translational Science
Institute, The Scripps Research Institute, La Jolla, California, United States of America, 3 Department of
Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, United
States of America
☯ These authors contributed equally to this work.
* gblander@insidetracker.com
Abstract
Exercise is effective toward delaying or preventing chronic disease, with a large body of evi-
dence supporting its effectiveness. However, less is known about the specific healthspan-
promoting effects of exercise on blood biomarkers in the disease-free population. In this
work, we examine 23,237 generally healthy individuals who self-report varying weekly run-
ning volumes and compare them to 4,428 generally healthy sedentary individuals, as well
as 82 professional endurance runners. We estimate the significance of differences among
blood biomarkers for groups of increasing running levels using analysis of variance
(ANOVA), adjusting for age, gender, and BMI. We attempt and add insight to our observa-
tional dataset analysis via two-sample Mendelian randomization (2S-MR) using large inde-
pendent datasets. We find that self-reported running volume associates with biomarker
signatures of improved wellness, with some serum markers apparently being principally
modified by BMI, whereas others show a dose-effect with respect to running volume. We fur-
ther detect hints of sexually dimorphic serum responses in oxygen transport and hormonal
traits, and we also observe a tendency toward pronounced modifications in magnesium sta-
tus in professional endurance athletes. Thus, our results further characterize blood biomark-
ers of exercise and metabolic health, particularly regarding dose-effect relationships, and
better inform personalized advice for training and performance.
Introduction
Physical inactivity is one of the leading modifiable behavioral causes of death in the US [1].
Worldwide, physical inactivity is estimated to account for about 8.3% of premature mortality,
an effect size that is on the same order as smoking and obesity [2]. At the same time, the potent
health benefits of exercise have been proven time and time again, with results so consistent
across a wide variety of chronic diseases that some posit it can be considered a medical inter-
vention [3–5]. However, since most investigators report the effects of exercise in either
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OPEN ACCESS
Citation: Nogal B, Vinogradova S, Jorge M,
Torkamani A, Fabian P, Blander G (2023) Dose
response of running on blood biomarkers of
wellness in generally healthy individuals. PLoS ONE
18(11): e0293631. https://doi.org/10.1371/journal.
pone.0293631
Editor: Efrem Kentiba, Arba Minch College of
Education, ETHIOPIA
Received: August 9, 2023
Accepted: October 16, 2023
Published: November 15, 2023
Peer Review History: PLOS recognizes the
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https://doi.org/10.1371/journal.pone.0293631
Copyright: © 2023 Nogal et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All Mendelian
Randomization data required to replicate the causal
analysis can be freely accessed at: https://gwas.
mrcieu.ac.uk/. No special access is required and all
diseased populations or athletes [6, 7], there exists a significant gap in knowledge as to the
measurable effects of exercise in the generally healthy population who exercise for the purpose
of improving their healthspan, which can be projected via established measures such as blood
biomarkers [8–11].
It is well established that routine laboratory biomarkers are validated proxies of the state of
an individual’s overall metabolic health and other healthpan-related parameters [12]. A large
body of evidence supports the effectiveness of exercise in modifying blood biomarkers toward
disease mitigation in clinical cohorts as well as athletes, where the effect sizes may be larger [6,
13]. Indeed, it’s been shown that more favorable changes in response to exercise training occur
usually in those with more pronounced dyslipidemia [13]. In professional athletes, the sheer
volume and/or intensity of physical activity may drive large effects in various hematological,
lipid, immune, and endocrine variables [6]. Our aim is to help fill the gap in understanding of
the effects of exercise on blood biomarkers in the generally healthy, free-living population.
Toward this end, we endeavored to explore the effects of vigorous exercise such as running in
apparently healthy, mostly non-athletic cohort to better understand the landscape of blood
biomarker modifications expected in the individual who partakes in recreational physical
activity for the purpose of maintaining good health.
For this purpose, we leveraged the InsideTracker dataset that includes information on self-
reported exercise habits combined with blood biomarker and genomics data. We have previ-
ously reported on the results of a longitudinal analysis on blood biomarker data from 1032
generally healthy individuals who used our automated, web-based personalized nutrition and
lifestyle platform [14]. For the purpose of this investigation, we focused on running as the exer-
cise of choice as it is one of the most common (purposeful) physical activity modalities prac-
ticed globally by generally healthy individuals and would thus be relevant. Moreover, since this
was a cross-sectional study based on self-reported exercise habits, we attempted to increase
our capacity to infer intervention effects, as well as tease out potential confounders, by per-
forming 2S-MR in large independent cohorts.
Materials and methods
Dataset
We conducted an observational analysis of data from InsideTracker users. InsideTracker is a
direct-to-consumer (DTC) company established in 2009 that markets and sells InsideTracker
(insidetracker.com), a personalized lifestyle recommendation platform. The platform provides
serum biomarker and genomics testing, and performs integrative analysis of these datasets,
combined with activity/sleep tracker data toward biomarker and healthspan optimization (of
note, at the time of this analysis, we did not have sufficient users with activity/sleep tracker
data to include this data stream in the current study). New users were continuously added to
the InsideTracker database from January 2011 to March 2022.
Recruitment of participants
Recruitment of participants aged between 18 and 65 and residing in North America was con-
ducted through company marketing and outreach. Participants were subscribing members to
the InsideTracker platform and provided informed consent to have their blood test data and
self-reported information used in an anonymized fashion for research purposes. Research was
conducted according to guidelines for observational research in tissue samples from human
subjects. Eligible participants completed a questionnaire that included age, ethnicity, sex, die-
tary preferences, physical activity, and other variables. This study employed data from 23,237
participants that met our analysis inclusion requirements, namely absence of any chronic
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the datasets used can be further freely accessed
via the free "TwoSampleMR" R package as
described in the methods (biomarker dataset
codes are shared in Supplementary tables as well).
The minimal dataset required to replicate the blood
biomarker results has been uploaded as
Supporting information.
Funding: InsideTracker was the sole funding
source. The funder provided support in the form of
salaries for authors B.N., S.V., P.F., M.J., and G.B.,
and was involved in the decision to publish, but did
not have an impact on the experimental design,
data analysis, and conclusions.
Competing interests: B.N., S.V., P.F., and G.B. are
employees of InsideTracker. This does not alter our
adherence to PLOS ONE policies on sharing data
and materials.
disease as determined by questionnaire and metabolic blood biomarkers within normal clini-
cal reference ranges. The platform is not a medical service and does not diagnose or treat med-
ical conditions, so medical history and medication use were not collected. The Institutional
Review Board (IRB) determine this work was not subject to a review based on category 4
exemption (“secondary research” with de-identified subjects).
Biomarker collection and analysis
Blood samples were collected and analyzed by Clinical Laboratory Improvement Amendments
(CLIA)–approved, third-party clinical labs (primarily Quest Diagnostics and LabCorp). Partic-
ipants were instructed to fast for 12 hours prior to the phlebotomy, with the exception of water
consumption. Results from the blood analysis were then uploaded to the platform via elec-
tronic integration with the CLIA-approved lab. Participants chose a specific blood panel from
7 possible offerings, each comprising some subset of the biomarkers available. Due to the vari-
ation in blood panels offered, the participant sample size per biomarker is not uniform.
Biomarker dataset preparation
In our raw dataset, occasional outlier values were observed that were deemed implausible (e.g.
fasting glucose < 65 mg/dL). To remove anomalous outliers in a systematic way, we used the
Interquartile Range (IQR) method of identifying outliers, removing data points which fell
below Q1–1.5 IQR or above Q3 + 1.5 IQR. The cohort was divided into five groups: profes-
sional endurance runners (PRO), high volume amateur (>10 h/week, HVAM), medium vol-
ume amateur (3–10 h/week, MVAM), low volume amateur (<3 h/week, LVAM), and
sedentary (SED).
Calculation of polygenic scores
The variants (SNPs) comprising the polygenic risk scores were derived from publicly available
GWAS summary statistics (https://www.ebi.ac.uk/gwas/). Scores were calculated across users
by summing the product of effect allele doses weighted by the beta coefficient for each SNP, as
reported in the GWAS summary statistics. Variant p-value thresholds were generally chosen
based on optimization of respective PGS-blood biomarker correlation in the entire Inside-
Tracker cohort with both blood and genomics datasets (~1000–1500 depending on the blood
biomarker at the time of analysis). Genotyping data was derived from a combination of a cus-
tom InsideTracker array and third party arrays such as 23andMe and Ancestry. Not all variants
for any particular PGS were genotyped on every array; proxies for missing SNPs were extracted
via the “LDlinkR” package using the Utah Residents (CEPH) with Northern and Western
European ancestry (CEU) population (R2 > 0.8 cut-off). Only results PGSs for which there
was sufficient biomarker-genotyping dataset overlap were reported (note that none of the
blood biomarker PGSs met this requirement).
Blood biomarker analysis with respect to running volume and polygenic
scores
To estimate significance of differences for blood biomarkers levels among exercise groups, we
performed 3-way analysis of variance (ANOVA) analysis adjusting for age, gender, and BMI
(type-II analysis-of-variance tables function ANOVA from ‘car’ R package, version 3.0–12).
When estimating the effort of reported training volume on biomarkers, we assigned numerical
values corresponding to 4 levels of running and performed ANOVA analysis with those levels
treating it as an independent variable. P-values were adjusted using the Benjamini & Hochberg
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method [15]. P-values for interaction plots were calculated with ANOVA including interaction
between exercise group and polygenic scores category. When comparing runners (PRO and
HVAM combined) versus sedentary individuals, we used propensity score matching method
to account for existing covariates (age and gender): we identified 745 sedentary individuals
with similar to runners’ age distributions among both males and females. We used ‘MatchIt’ R
package (version 4.3.3) implementing nearest neighbor method for matching [16].
Mendelian randomization
We attempted to add insight around the causality of exercise vs. BMI differences with respect
to serum marker improvement by performing MR analyses on a subset of biomarker observa-
tions where BMI featured as a strong covariate and was thus used as the IV in the 2S-MR.
Thus, our hypothesis here was that BMI differences were the primary (causal) driver behind
the improvement behind some biomarkers. MR uses genetic variants as modifiable exposure
(risk factor) proxies to evaluate causal relationships in observational data while reducing the
effects of confounders and reverse causation (S1 Fig in S1 File). These SNPs are used as instru-
mental variables and must meet 3 basic assumptions: (1) they must be robustly associated with
the exposure; (2) they must exert their effect on outcome via the exposure, and (3) there must
be no unmeasured confounders of the associations between the genetic variants and outcome
(e.g. horizontal pleiotropy) [17]. Importantly, SNPs are proper randomization instruments
because they are determined at birth and thus serve as proxies of long-term exposures and can-
not, in general, be modified by the environment. If the 3 above mentioned assumptions hold,
MR-estimate effects of exposure on outcomes are not likely to be significantly affected by
reverse causation or confounding. In the 2S-MR performed here, where GWAS summary sta-
tistics are used for both exposure and outcome from independent cohorts, reverse causation
and horizontal pleiotropy can readily be assessed, and weak instrument bias and the likelihood
of false positive findings are minimized as a result of the much larger samples sizes [17].
Indeed, the bias in the 2S-MR using non-overlapping datasets as performed here is towards
the null [17]. Furthermore, to maintain the SNP-exposure associations and linkage disequilib-
rium (LD) patterns in the non-overlapping populations we used GWAS datasets from the
MR-Base platform that were derived from ancestrally similar populations (“ukb”: analysis of
UK Biobank phenotypes, and “ieu”: GWAS summary datasets generated by many different
European consortia). To perform the analysis we used the R package “TwoSampleMR” that
combines the effects sizes of instruments on exposures with those on outcomes via a meta-
analysis. We used “TwoSampleMR” package functions for allele harmonization between expo-
sure and outcome datasets, proxy variant substitution when SNPs from exposure were not
genotyped in the outcome data (Rsq>0.8 using the 1000G EUR reference data integrated into
MR-Base), and clumping to prune instrument SNPs for LD (the R script used for MR analyses
is available upon request). We used 5 different MR methods that were included as part of the
“TwoSampleMR” package to control for bias inherent to any one technique [18]. For example,
the multiplicative random effects inverse variance-weighted (IVW) method is a weighted
regression of instrument-outcome effects on instrument-exposure effects with the intercept is
set to zero. This method generates a causal estimate of the exposure trait on outcome traits by
regressing the, for example, SNP-BMI trait association on the SNP-biomarker measure associ-
ation, weighted by the inverse of the SNP-biomarker measure association, and constraining
the intercept of this regression to zero. This constraint can result in unbalanced horizontal
pleiotropy whereby the instruments influence the outcome through causal pathways distinct
from that through the exposure (thus violating the second above-mentioned assumption).
Such unbalanced horizontal pleiotropy distorts the association between the exposure and the
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outcome, and the effect estimate from the IVW method can be exaggerated or attenuated.
However, unbalanced horizontal pleiotropy can be readily assessed by the MR Egger method
(via the MR Egger intercept), which provides a valid MR causal estimate that is adjusted for
the presence of such directional pleiotropy, albeit at the cost of statistical efficiency. Finally, to
ascertain the directionality of the various causal relationships examined, we also performed
each MR analysis in reverse where possible.
Results
Study population characteristics
Table 1 shows the demographic characteristics of the study population. We observed a signifi-
cant trend toward younger individuals reporting higher running volume, with more than 75%
of the professional (PRO) group falling between the ages of 18 and 35 (S1 Table in S1 File). Sig-
nificant differences were also observed in the distribution of males and females within study
groups (Table 1). Moreover, higher running volume associated with significantly lower body
mass index (BMI). Thus, moving forward, combined comparisons of blood biomarkers as they
relate to running volume were adjusted for age, gender, and BMI.
Endurance exercise exhibits a modest association with clusters of blood
biomarker features
In order to begin to understand the most important variables that may associate with endur-
ance exercise in the form of running, we performed a principal component analysis (PCA),
sub-dividing the male and female cohorts into two most divergent groups in terms of exercise
volume: PRO/high volume amateur (HVAM) and sedentary (SED) groups. Using propensity
matching, PRO and amateur athletes who reported running >10h per week were combined
into the PRO-HVAM group to balance out the sample size between the exercising and non-
exercising groups. This approach yielded a modest degree of separation, with hematological,
inflammation, and lipid features, as well as BMI explaining some of the variance (Fig 1A
through 1D). We hypothesized that there may more subtle relationships between running vol-
ume and the blood biomarker features that contributed to distinguishing the endurance exer-
cise and sedentary groups, thus we next performed ANOVA analyses stratified by running
volume as categorized in Table 1.
Significant trends in glycemic, hematological, blood lipid, and
inflammatory serum traits with increasing running volumes
Weighted ANOVA analyses adjusted for age, gender, and BMI showed significant differences
among groups for multiple blood biomarkers (Table 2 and S2 Table in S1 File, Figs 2 and 3).
Table 1. Study population demographics.
Group
N
Female, %
Age, yrs
Body mass index, kg/m2
PRO
82
53.7%
33.68
20.15 ± 6.02
HVAM
1103
52.9%
39.48
22.57 ± 9.97
MVAM
6747
54.2%
41.49
23.35 ± 9.76
LVAM
10877
34.2%
41.16
24.72 ± 9.70
SED
4428
48.9%
44.25
27.83 ± 10.70
PRO = Professional, HVAM = high volume amateur (>10 h/week), MVAM = medium volume amateur (3–10 h/week), LVAM = low volume amateur (<3 h/week),
SED = sedentary
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We observed a trend toward lower HbA1c, hsCRP, RDW, WBC, ferritin, gamma-glutamyl
transferase (GGT), and LDL. HDL, hemoglobin (Hb), transferrin saturation (TS), alanine ami-
notransferase (ALT), aspartate aminotransferase (AST), vitamin B12, folate, 25-hydroxy vita-
min D, and creatine kinase (CK) tended to be higher with increasing reported training
volume, particularly in PRO runners (Table 2 and S2 Table in S1 File, Figs 2 and 3 and S2 Fig
in S1 File). Hct and Hb were higher only in PRO males, whereas increased running volume
associated with upward trend in these biomarkers in females (Fig 3A and 3B). Increased run-
ning volume was associated with markedly lower Fer in males, whereas female runners did not
exhibit varying levels, and SED females showed increased levels (Fig 3C). The low ferritin
observed in male and female runners was not clinically significant. ALT positively associated
with running volume in females only (S2 Fig in S1 File). Serum and RBC magnesium (Mg)
were both significantly lower in PRO runners relative to all other groups (Table 2 and Fig 3D
Fig 1. Principal component analysis and variables plots of PRO-HVAM runners and sedentary user blood biomarkers. Females, (A) and (B); males
(C) and (D). PRO-HVAM = combined professional and high-volume amateur. Alb = albumin, ALT = alanine transaminase, AST = aspartate
aminotransferase, B12 = vitamin B12, Ca = calcium, Chol = total cholesterol, CK = creatine kinase, Cor = cortisol, FE = iron, EOS_PCT = eosinophil
percentage, Fer = ferritin, Fol = folate, FT = free testosterone, GGT = gamma-glutamyl transferase, Glu = glucose, Hb = hemoglobin,
HCT = hematocrit, HDL = high density lipoprotein, HbA1c = glycated hemoglobin, hsCRP = high-sensitivity C-reactive protein, LDL = low density
lipoprotein, LYMPS_PCT = lymphocyte percentage, MCH = mean cell hemoglobin, Mg = magnesium, MONOS_PCT = monocytes percentage,
MPV = mean platelet volume, Na = sodium, RBC = red blood cells, RBC_Mg = red blood cell magnesium, RDW = red blood cell distribution width,
SHBG = sex hormone binding globulin, Tg = triglycerides, TIBC = total iron binding capacity, WBC = white blood cells.
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and 3E). Increasing levels of endurance exercise also appeared to be associated with higher
sex-hormone binding globulin (SHBG), particularly in PRO male runners (Fig 3F).
Endurance exercise correlates with lower BMI across categories of genetic
risk
Using publicly available GWAS summary statistics, we constructed blood biomarker polygenic
risk scores (PGSs) to explore potential genetic risk-mitigating effects of endurance exercise.
Since only a subset of the individuals in our cohort were genotyped, we aggregated the groups
into 2 categories—PRO-HVAM and sedentary—to increase statistical power. This across-
group sample size increase generally did not sufficiently power the ANOVA analysis to detect
statistically significant trends, though the BMI polygenic risk was suggestively mitigated for
both males and female PRO-HVAM runners across categories of genetic risk (Fig 4B).
Increased running volume is associated with lower BMI which may drive
biomarker changes
We observed a significant downward trend in the BMI with increased running volume for
both males and females, and, although some of the biomarker differences between sedentary
Table 2. Blood biomarkers significantly different among sedentary individuals and those who partake in running for exercise to various degrees.
BIOMARKER
ANOVA P-VALUE
TREND P-VALUE
LOWEST MEAN
HIGHEST MEAN
ALB
<1e-16
<0.001
MVAM
PRO
ALT
<1e-16
<1e-16
SED
PRO
AST
<1e-16
<0.001
SED
PRO
B12
<0.001
<0.001
SED
PRO
CHOL
<0.001
0.005
PRO
SED
CK
<1e-16
<1e-16
SED
PRO
COR
<0.001
0.675
SED
PRO
FE
<0.001
0.119
SED
PRO
FER
<1e-16
<1e-16
MVAM
SED
FOL
<1e-16
<0.001
SED
PRO
FT
<0.001
0.013
SED
PRO
GGT
<1e-16
<0.001
PRO
SED
GLU
0.087
0.184
PRO
SED
HB
0.002
<0.001
MVAM
PRO
HCT
0.053
0.055
MVAM
PRO
HDL
<1e-16
<0.001
SED
PRO
HBA1C
<0.001
0.010
PRO
SED
HSCRP
<0.001
0.176
PRO
SED
LDL
<0.001
0.006
PRO
SED
MG
<0.001
0.276
PRO
SED
MPV
0.058
0.089
SED
HVAM
NA
<1e-16
0.622
HVAM
SED
RBC_MG
<0.001
0.773
PRO
SED
RDW
<1e-16
0.002
PRO
SED
SHBG
<1e-16
0.004
SED
PRO
TG
<1e-16
<1e-16
PRO
SED
WBC
<1e-16
<1e-16
PRO
SED
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and exercising individuals remained significant after adjustment for BMI, their significance
was attenuated (Fig 4A). Thus, we hypothesized that BMI may be driving a significant portion
of the observed variance in some of the biomarkers across the groups. Thus, to explore causal
relationships between weight and biomarker changes, we performed 2S-MR with BMI-associ-
ated single-nucleotide polymorphisms (SNPs) as the instrumental variables (IVs) for a subset
of the healthspan-related biomarkers where BMI explained a relatively large portion of the
Fig 2. Blood biomarkers associated with running: Inflammation proxies, (A) hsCRP = high-sensitivity C-reactive protein and (B) WBC = white blood
cells; blood lipids, (C) HDL = high density lipoprotein (D) LDL = low density lipoprotein, and (E) Tg = triglycerides; glycemia proxies, (F)
Glu = glucose and (G) HgbA1c = glycated hemoglobin, and (H) Cor = cortisol.
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Fig 3. Blood biomarkers associated with running: (A and B) Hb (hemoglobin) and Hct (hematocrit) increase with increasing running volume, (C) Fer
(ferritin) is reduced with increasing running volume, (D and E) Serum and RBC Mg (red blood cell magnesium) are reduced in professional runners,
and (F) SHBG (sex hormone binding globulin) levels increase with increasing running volume in males.
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Fig 4. BMI significantly varied among running groups (A) with some suggestive effects on BMI PGS modification (total number for observations
(N) for T1, T2, and T3 were 87, 84, and 100, respectively) (B) T1, T2, and T3 = 1st, and 2nd and 3rd tertials of the polygenic score distribution.
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variance in our analysis. In general, these blood biomarkers associated with inflammation
(hsCRP and RDW), lipid metabolism (Tg and HDL), glycemic control (HbA1c and Glu), as
well as Alb and SHBG. We used GWAS summary statistics and found that most of these BMI-
blood biomarker relationships examined directionally aligned with our study (except for
LDL), and some were indicative of causal relationships in the BMI-biomarker direction even
after considering directional pleiotropy (S3 Table in S1 File). We entertained the possibility of
reverse causality and thus repeated the 2S-MR using each of the biomarker levels as the expo-
sure and BMI as the outcome, and the results were generally not significant (except for WBC–
see S4 Table in S1 File). Of note, to estimate the direct causal effects of running on blood
parameters, we attempted to find an instrumental variable for to approximate running as the
exposure from publicly available GWAS summary statistics. Toward this end, we found that
increasing levels of vigorous physical activity did associate with lower hsCRP, HbA1C, higher
HDL, and possibly higher SHBG (although the explained variance (R2) in this exposure was
just 0.001009, the F statistic was 37.7, thus meeting the criteria of F > 10 for minimizing weak
instrument bias) (Fig 5 and S3 Fig in S1 File; S5 Table in S1 File).
Vigorous physical activity associates with healthier behaviors
We hypothesized that those who exercise regularly may also partake in other healthful lifestyle
habits that may be contributing to more optimal blood biomarker signatures of wellness. How-
ever, our dataset did not allow for systematic accounting of other lifestyle habits across all run-
ning groups. Thus, we again leveraged the potential of the 2S-MR approach to inform
potential confounding associations between modifiable exposures and found that vigorous
physical activity such as running is at least suggestively associated with several behaviors asso-
ciated with improved health (S4 Fig in S1 File). Our analysis showed that those who participate
in increasing levels of vigorous physical activity may be less likely to eat processed meat (IVW
p = 0.0000013), sweets (IVW p = 0.32), and nap during the day (IVW p = 0.13), while increas-
ing their intake of oily fish (IVW p = 0.029), salad/raw vegetable intake (IVW p = 0.00016),
and fresh fruit (IVW p = 0.0027) (S6 Table in S1 File). Furthermore, following our assessment
Fig 5. Two-sample Mendelian randomization shows that increasing levels of vigorous physical activity such as running is associated with
improvement of (A) hsCRP = high-sensitivity C-reactive protein, (B) HDL = high density lipoprotein, and (C) HbA1c = glycated
hemoglobin levels.
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of reverse causality, we found evidence for the bidirectionality in the causal relationship
between vigorous activity and napping during the day and salad/raw vegetable intake, perhaps
suggesting some degree of confounding due to population stratification (S7 Table in S1 File).
The suggestive positive effect of fresh fruit and processed meat intake on vigorous physical
activity appeared to violate MR assumption (3) (S1 Fig in S1 File) (horizontal pleiotropy p-val-
ues 0.051 and 0.17, respectively–S5 Fig in S1 File).
Discussion
In this report, we describe the variance in wellness-related blood biomarkers among self-
reported recreational runners, PRO runners, and individuals who do not report any exercise.
Overall, we find that 1) recreational running as an exercise appears to be an effective interven-
tion toward modifying several biomarkers indicative of improved metabolic health, 2) an
apparent dose-response relationship between running volume and BMI may itself be responsi-
ble for a proportion of the apparent metabolic benefits, and 3) both PRO-level status and gen-
der appear to associate with heterogeneous physiological responses, particularly in iron and
magnesium metabolism, as well as some hormonal traits.
Self-reported running improves glycemia and lipidemia
We did not observe distinct clusters corresponding to self-reported high-volume/PRO runners
and the sedentary upon dimension reduction. This is, perhaps, not unexpected due, in part, to
the self-selected healthspan-oriented nature of our cohort, where even the sedentary subset of
individuals tends to exhibit blood biomarker levels in the normal clinical reference ranges.
Furthermore, the measurement of running volume via self-report may be vulnerable to overes-
timation, which may have contributed to the blending of sedentary and exercise groups with
respect to the serum markers measured, resulting in only marginal separation between the
groups [19, 20]. However, we did observe significant individual blood biomarker variance
with respect to reported running volumes when the dataset was subjected to ANOVA, even
after adjustment for age, sex, and BMI.
From among glycemic control blood biomarkers, we were able to detect a relatively
small exercise effect in both fasting glucose and HbA1c in this generally healthy cohort,
where the average measures of glycemia were below the prediabetic thresholds in even the
sedentary subset of the cohort. Larger exercise intervention effects on metabolic biomarkers
may be expected in cohorts that include individuals with more clinically significant baseline
values [21].
Similarly, blood lipids improved with higher self-reported running volume, and this result
has been reported before in multiple controlled endurance exercise trials [22]. The literature
indicates that HDL and Tg are two exercise-modifiable blood lipid biomarkers, with HDL
being the most widely reported to be modified by aerobic exercise [23, 24]. Although the
mechanism behind this is not entirely clear, it likely involves the modification of lecithin
acyltransferase and lipoprotein lipase activities following exercise training [25]. We observed
a similar trend in our blood biomarker analysis, with HDL exhibiting an upward trend with
increasing reported running volume. While we also found Tg and LDL to decrease with
increasing exercise volume, these trends were less pronounced. Reports generally suggest
that, in order to reduce LDL more consistently, the intensity of aerobic exercise must be high
enough [23]. In the case of Tg, baseline levels may have a significant impact on the exercise
intervention effect, with individuals exhibiting higher baselines showing greater improve-
ments [13].
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Importantly, these results suggests that exercise has a significant effect on glycemic control
and blood lipids even in the self-selected, already healthy individuals who are proactive about
preventing cardiometabolic disease.
Self-reported running and serum proxies of systemic inflammation
Chronic low-grade inflammation is one of the major risk factors for compromised cardiovas-
cular health and metabolic syndrome (MetS). While there is no shortage of inflammation-
reducing intervention studies on CVD patients with clinically high levels of metabolic inflam-
mation, there is less emphasis on modifiable lifestyle factors that can help stave off CVD and
extend healthspan in the generally healthy individual. Indeed, considering the pathological
cardiovascular processes begin shortly after birth, prevention in asymptomatic individuals
may be a more appropriate strategy toward decreasing the burden of CVD on the healthcare
system [26].
Toward this end, increasing self-reported running volume appeared to associate with
improved markers of inflammation, as shown by the lower levels of hsCRP, WBC, as well as
ferritin. Of note, while the acute-phase protein, ferritin, is often used in the differential diagno-
sis of iron deficiency anemia, the biomarker’s specificity appears to depend on the inflamma-
tory state of the individual, as it associates with hsCRP and inflammation more than iron
stores, particularly in those with higher BMI [27]. Although serum ferritin and iron is reported
to be lower in male and female elite athletes [28], the observed overall negative association of
ferritin with increased running volume in our cohort may be an indication of lower levels of
inflammation rather than compromised iron stores, particularly since the average ferritin level
across all groups was above the clinical iron deficiency thresholds. Moreover, increased levels
of ferritin have been associated with insulin resistance and lower levels of adiponectin in the
general population, both indicators of increased systemic inflammation [29]. Here, exercising
groups with lower levels of ferritin also exhibited glycemic and blood lipid traits indicative of
improved metabolic states, further supporting ferritin’s role as an inflammation proxy. Finally,
Hb, TS and iron tended to be higher in those who run for exercise compared to the SED group
(with the TIBC lower), again suggesting that runners, including the PRO group, were iron-
sufficient in this cohort.
PRO endurance runners exhibit distinct biomarker signatures
PRO athletes exhibited lower serum and RBC Mg, which may be indication of the often-
reported endurance athlete hypomagnesaemia [30]. While the serum Mg was still within nor-
mal clinical reference range for both PRO female and male athletes, RBC Mg, a more sensitive
biomarker of Mg status [31], was borderline low in female PRO athletes and might suggest
suboptimal dietary intakes and/or much higher volume of running training compared to the
other running groups (i.e. >>10h /week). Indeed, this group also had elevated baseline CK
and AST, which suggests a much higher training intensity and/or volume. Moreover, PRO
level athletes had adequate iron status and serum B12 and folate in the upper quartile of the
normal reference range, suggesting that these athletes’ general nutrition status may have been
adequate. These observations suggest that elite endurance runners may need to pay particular
attention to their magnesium status.
Further, we observed higher levels of SHBG in PRO male runners, a biomarker whose levels
positively correlate with various indexes of insulin sensitivity [32]. However, since the average
SHBG levels in the SED group were not clinically low in both sexes, the observed increase in
SHBG levels induced by running in males may be a catabolic response, as cortisol levels in this
group were also higher. Indeed, Popovic et al. have shown that endurance exercise may
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increase SHBG, cortisol, and total testosterone levels at the expense of free testosterone levels
[33]. This could perhaps in part be explained by higher exercise-induced adiponectin levels,
which have been shown to increase SHBG via cAMP kinase (AMPK) activation [34]. However,
since our data is observational, we cannot rule out overall energy balance as a significant con-
tributor to SHBG levels. For example, caloric restriction (CR) has been shown to result in
higher SHBG and cortisol levels [32].
Finally, regarding the abovementioned PRO group elevated AST and CK biomarkers, evi-
dence suggests that normal reference ranges in both CK and AST in well-recovered athletes
should be adjusted up, as training and competition have a profound, non-pathological, impact
on the activity of these enzymes [35, 36]. Indeed, the recommendation appears to be not to use
reference intervals derived from the general population with hard-training (particularly com-
petitive) athletes [36].
Effect of BMI on blood biomarkers
Since the current study is a cross-sectional analysis of self-reported running, we could not rule
out the possibility that factors other than exercise were the driving force behind the observed
biomarker variance among the groups examined. These factors, such as diet, sleep, and/or
medications were not readily ascertained in this free-living cohort at the time of this study, but
BMI was readily available to evaluate this biomarker’s potential relative contribution to the
observed mean biomarker differences among self-reported runner groups.
Multiple studies have attempted to uncouple the effects of exercise and BMI reduction on
blood biomarker outcomes, with mixed results [37]. For example, it is relatively well-known
that acute bouts of exercise improve glucose metabolism, but long-term effects are less well
described [38]. Indeed, whether exercise without significant weight-loss is effective toward
preventing metabolic disease (and the associated blood biomarker changes) is inconclusive
[39–41]. From the literature, it appears that, for endurance exercise to have significant effect
on most blood biomarkers, the volume of exercise needs to be very high, and this typically
results in significant reduction in weight. Thus, in practice, it is difficult to demonstrably
uncouple the effects of significant exercise and the associated weight-loss, and the results
may depend on the blood biomarker in question. Indeed, there is evidence that exercise
without weight-loss does improve markers of insulin sensitivity but not chronic inflamma-
tion, with the latter apparently requiring a reduction in adiposity in the general population
[39–41].
In our study of apparently healthy individuals, we observed a downward trend in BMI with
increasing self-reported running volume, and, although this study was not longitudinal and we
are thus unable to claim weight-loss, our 2S-MR analysis using BMI as the exposure nonethe-
less suggests this biomarker to be responsible for a significant proportion of the modification
of some blood biomarkers.
Serum markers of systemic inflammation.
Through our 2S-MR analyses, we show that
BMI is causally associated with markers of systemic inflammation, including RDW, folate,
and hsCRP [27, 42, 43]. Similar analyses have reported that genetic variants that associate
with higher BMI were associated with higher CRP levels, but not the other way around [44].
The prevailing mechanism proposed to explain this relationship appears to be the pathologi-
cal nature of overweight/obesity-driven adipose tissue that results in secretion of proinflam-
matory cytokines such as IL-6 and TNFa, which then stimulate an acute hepatic response,
resulting in increased hsCRP levels (among other effects) [45]. Thus, our 2S-MR analyses
and those of others [44] would indicate that the primary factor behind the lower systemic
inflammation in our cohort may be the exercise-associated lower BMI and not running
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exercise per se, though the lower hsCRP in runners remained significant after adjustment for
BMI in our analysis.
Indeed, although a major driver behind reduced systemic inflammation may be a reduction
in BMI in the general population, additive effects of other lifestyle factors such as exercise can-
not be excluded. For example, a large body of cross-sectional investigations does indicate that
physically active individuals exhibit CRP levels that are 19–35% lower than less active individu-
als, even when adjusted for BMI as was the case in the current analysis [41]. Further, it’s been
reported that physical activity at a frequency of as little as 1 day per week is associated with
lower CRP in individuals who are otherwise sedentary, while more frequent exercise further
reduces inflammation [41].
Significantly, our entire cohort of self-selected apparently healthy individuals did not
exhibit clinically high hsCRP, with average BMI also below the overweight thresholds. Because
all subjects were voluntarily participating in a personalized wellness platform intended to opti-
mize blood biomarkers that included hsCRP, it is possible that some individuals from across
the study groups (both running and sedentary) in our cohort partook in some form of inflam-
mation-reducing dietary and/or lifestyle-based intervention. Thus, that we detected a signifi-
cant difference in hsCRP between exercising and non-exercising individuals in this self-
selected already generally healthy cohort may be suggestive of the potential for additional pre-
ventative effect of scheduled physical activity on low-grade systemic inflammation in the gen-
erally healthy individual.
Blood lipids.
Controlled studies that tightly track exercise and the associated adiposity
reduction have reported that body fat reduction (and not improvement in fitness as measured
via VO2max) is a predictor of HDL, LDL, and Tg [46]. Similarly, though BMI is an imperfect
measure of adiposity, our 2S-MR analysis suggests that this biomarker is causally associated
with improved levels of HDL and Tg, though not LDL. This latter finding replicates a report
by Hu et al. who, using the Global Lipids Genetics Consortium GWAS summary statistics,
applied a network MR approach that revealed causal associations between BMI and blood lip-
ids, where Tg and HDL, but not LDL, were found to trend toward unhealthy levels with
increasing adiposity [47]. On the other hand, others implemented a robust BMI genetic risk
score and demonstrated a causal association of adiposity with peripheral artery disease and a
multiple linear regression showed a strong association with HDL, TC, and LDL, among other
metabolic parameters [48]. In our cohort, given the lack of evidence for a causal BMI-LDL
association and the overall healthy levels of BMI, the observed a significant improvement in
LDL may be a result of marked running intensity and/or volume, possibly combined with the
aforementioned additional wellness program intervention variables.
Hormonal traits.
As described above, we observed a trend toward increased plasma corti-
sol and SHBG in runners, particularly PRO level athletes. The effects on cortisol are consistent
with a report by Houmard et al., who found male distance runners to exhibit higher levels of
baseline cortisol [49]. With respect to the effects of BMI on baseline cortisol levels, this obser-
vation is generally supported by our 2S-MR analyses with evidence for a consistent effect of
increased cortisol with decreasing BMI. However, this association was suggestive at best, indi-
cating that the higher levels of cortisol exhibited in the PRO runners with significant lower adi-
posity are not likely to be solely explained by their lower BMI. Indeed, the relationship
between BMI and cortisol appears to be complex, with some reports suggesting a U-shaped
relationship, where the glucocorticoid’s levels associate negatively up to about a BMI of 30 kg/
m2, then exhibiting a positive correlation into obesity phenotypes [50]. MR statistical models
generally do not account for such non-linearity and would require a more granular demo-
graphical treatment, which is not possible using only GWAS summary statistics data in the
context of 2S-MR [17, 51].
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Behavioral traits associated with increase physical activity
The combination of the body of the literature that describes the effects of endurance training
on blood biomarkers, and our own analysis that included markers such as CK and AST, makes
us cautiously assured that most of the abovementioned blood biomarker signatures are indeed
a result of the interplay between self-reported running and the associated lower BMI. How-
ever, as this is a self-report-based analysis and we were unable to track other subject behaviors
in this free-living cohort, we acknowledge that multiple behaviors that associate with exercise
may be influencing our results.
Toward this end, our exploratory 2S-MR analyses revealed potentially causal relationships
between vigorous exercise and multiple dietary habits that have been shown to improve the
biomarkers we examined. Indeed, diets that avoid processed meat and sweets while providing
ample amounts of fresh fruits, as well as oily fish have been shown to be anti-inflammatory,
and improve glycemic control and dyslipidemia [52, 53]. That physically active individuals are
also more likely to make healthier dietary choices adds insight to the potential confounders in
ours and others’ observational analyses, and this similar associations have previously been
reported [54–56]. For example, using a calculated healthy eating motivation score, Naughton
et al. showed that those who partake in more than 2 hours of vigorous physical activity are
almost twice as likely to be motivated to eat healthy [56]. Indeed, upon closer examination, the
genetic instruments used to approximate vigorous physical activity as the exposure in this
work included variants in the genes DPY19L1, CADM2, CTBP2, EXOC4, and FOXO3 [57]. Of
these, CADM2 encodes proteins that are involved in neurotransmission in brain regions well
known for their involvement in executive function, including motivation, impulse regulation
and self-control [58]. Moreover, variants within this locus have been associated with obesity-
related traits [59]. Thus, it is likely that the improved metabolic outcomes seen here with our
self-reported runners are a composite result of both these individuals exercise and dietary hab-
its. Importantly, the above suggests that a holistic wellness lifestyle approach is in practice the
most likely to be most effective toward preventing cardiometabolic disease. Nonetheless, the
focus of this work–exercise in the form of running–is known to significantly improve cardiore-
spiratory fitness (CRF), which has been shown to be an independent predictor of CVD risk
and total mortality, outcomes that indeed correlate with dysregulated levels in many of the
blood biomarkers examined in this work [7].
Study limitations
This study is based on self-reported running and thus has several limitations. First, it is gener-
ally known that subjects tend to overestimate their commitment to exercise when self-report-
ing, although in our cohort is a self-selected health-oriented population that is possibly less
likely to over-report their running volume. Furthermore, although the robust increasing trend
in baselines for muscle damage biomarkers (CK, AST) that have been shown to be associated
with participation in sports and exercise provides indirect evidence that the running groups
were indeed participating in increasing volumes of strenuous physical activity, we cannot con-
firm whether the reported running was performed overground or on a treadmill, which may
result in some heterogeneity in physiological responses, nor can we ascertain the actual train-
ing volume of PRO-level runners. We also cannot exclude the possibility that the running
groups also participated in other forms of exercise (such as strength training) or partook in
other wellness program interventions that may have influenced their blood biomarkers and/or
BMI via lean muscle accretion. Toward this end, we have attempted to shed light on potential
behavioral covariates related to vigorous physical activity via 2S-MR. Finally, while this cohort
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is generally healthy, we cannot exclude the potential for unmeasured confounders such as
medications, nutritional supplements, and unreported health conditions.
2S- MR enables the assessment of causal relationships between modifiable traits and is less
prone to the so-called “winner’s curse” that more readily affects one-sample MR analyses [17,
51]. Because 2S-MR uses GWAS summary statistics for both exposure and outcome, it is possi-
ble to increase statistical power because of the increased sample sizes. However, horizontal
pleiotropy is still a concern that can skew the results. Currently, there is no gold standard MR
analysis method, thus we used different techniques (IVW, MR-Egger, and median-based esti-
mations–all of which are based on different assumptions and thus biases) to evaluate the con-
sistency among these estimators and only reported associations as ‘causal’ if there was cross-
model consistency. Nonetheless, an exposure such as BMI is a complex trait that is composed
of multiple sub-phenotypes (such as years of education) that could be driving the causal
associations.
Conclusions
Running is one of the most common forms of vigorous exercise practiced globally, thus mak-
ing it a compelling target of research studies toward understanding its applicability in chronic
disease prevention. Our cross-sectional study offers insight into the biomarker signatures of
self-reported running in generally healthy individuals that suggest improved insulin sensitivity,
blood lipid metabolism, and systemic inflammation. Furthermore, using 2S-MR in indepen-
dent datasets we provide additional evidence that some biomarkers are readily modified BMI
alone, while others appear to respond to the combination of varying exercise and BMI. Our
additional bi-directional 2S-MR analyses toward understanding the causal relationships
between partaking in vigorous physical activity and other healthy behaviors highlight the
inherent challenge in disambiguating exercise intervention effects in cross sectional studies of
free-living populations, where healthy behaviors such as exercising and healthy dietary habits
co-occur. Overall, our analysis shows that the differences between those who run and the sed-
entary in our cohort are likely a combination of the specific physiological effects of exercise,
the associated changes in BMI, and lifestyle habits associated with those who exercise, such as
food choices and baseline activity level. Looking ahead, the InsideTracker database is continu-
ously augmented with blood chemistry, genotyping, and activity tracker data, facilitating fur-
ther investigation of the effects of various exercise modalities on phenotypes related to
healthspan, including longitudinal analyses and more granular dose-response dynamics.
Supporting information
S1 File.
(PDF)
S1 Dataset.
(TXT)
Acknowledgments
We thank Michelle Cawley and Renee Deehan for their assistance with background subject
matter research and insightful conversations.
Author Contributions
Conceptualization: Bartek Nogal.
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Data curation: Svetlana Vinogradova.
Formal analysis: Bartek Nogal, Svetlana Vinogradova, Paul Fabian.
Investigation: Bartek Nogal, Svetlana Vinogradova.
Methodology: Bartek Nogal, Svetlana Vinogradova.
Project administration: Bartek Nogal.
Supervision: Milena Jorge, Ali Torkamani, Gil Blander.
Visualization: Svetlana Vinogradova.
Writing – original draft: Bartek Nogal.
Writing – review & editing: Bartek Nogal.
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