The Influence of Physical Activity on the Gut Microbiome of Pre-Diabetic Adults
Phase: NA
Conditions: Obesity, Overweight, Overweight and Obesity, PreDiabetes, Type 2 Diabetes, Type 2 Diabetes Mellitus, Type 2 Diabetes Mellitus in Obese
Sponsor: University of Minnesota

MEDICAL PROTOCOL (HRP-590) 
PROTOCOL TITLE: Exercise and the Gut Microbiome R21 
VERSION DATE: 05/11/2022 
 
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PROTOCOL COVER PAGE 
 
 
Protocol Title 
The Influence of Physical Activity on the Gut Microbiome of 
Pre-Diabetic Adults 
Principal 
Investigator/Faculty 
Advisor 
Name: Ryan T. Demmer, PhD, MPH & Mark Pereira, PhD, MPH  
Department:  School of Public Health, Division of Epidemiology 
and Community Health 
Telephone Number: (612)-626-8597 
Email Address: demm0009@umn.edu 
Student 
Investigator 
Name: N/A 
Current Academic Status (Student, Fellow, Resident): N/A  
Department: N/A 
Telephone Number: N/A 
Institutional Email Address: N/A 
Scientific 
Assessment 
Nationally-based, federal funding organizations 
IND/IDE # (if 
applicable) 
N/A. 
IND/IDE Holder 
N/A. 
Investigational Drug 
Services # (if 
applicable) 
N/A. 
Version 
Number/Date: 
Version 1: 02/28/2020 
Version 2: 03/25/2020 
Version 3: 04/14/2021 
Version 4: 05/25/2021 
Version 5: 07/21/2021 
Version 6: 09/08/2021 
Version 7: 05/11/2022 

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ANCILLARY REVIEWS 
Which ancillary reviews do I need and when do I need them? 
Refer to HRP-309 for more information about these ancillary reviews. 
Select yes or 
no 
Does your study… 
If yes… 
Impact on 
IRB Review 
☐ Yes 
☒ No 
 
Include Gillette resources, staff 
or locations 
Gillette Scientific review and Gillette 
Research Administration approval is 
required.  Contact: 
research@gillettechildrens.com   
Required 
prior to IRB 
submission 
☐ Yes 
☒ No 
Involve Epic, or Fairview 
patients, staff, locations, or 
resources? 
The Fairview ancillary review will be 
assigned to your study by IRB staff 
Contact: ancillaryreview@Fairview.org 
Approval 
must be 
received 
prior to IRB 
committee/ 
designated 
review.   
 
Consider 
seeking 
approval 
prior to IRB 
submission. 
☐ Yes 
☒ No 
Include evaluation of drugs, 
devices, biologics, tobacco, or 
dietary supplements or data 
subject to FDA inspection? 
The regulatory ancillary review will be 
assigned to your study by IRB staff 
Contact: medreg@umn.edu  
 
See: 
https://policy.umn.edu/research/indide  
☐ Yes 
☒ No 
Require Scientific Review? Not 
sure? See guidance on next 
page. 
 
Documentation of scientific merit must 
be provided.   
Contact: hrpp@umn.edu  
☐ Yes 
☒ No 
Relate to cancer patients, 
cancer treatments, cancer 
screening/prevention, or 
tobacco? 
Complete the CPRC application process.  
Contact: ccprc@umn.edu  
☐ Yes 
☒ No 
Include the use of radiation? 
(x-ray imaging, 
radiopharmaceuticals, external 
beam or brachytherapy) 
Complete the AURPC Human Use 
Application and follow instructions on 
the form for submission to the AURPC 
committee. 
Contact: barmstro@umn.edu  
Approval 
from these 
committees 
must be 
received 
prior to IRB 
approval;  
 
These 
groups 
each have 
their own 
application 
process.  
☐ Yes 
☒ No 
Use the Center for Magnetic 
Resonance Research (CMRR) 
as a study location? 
Complete the CMRR pre-IRB ancillary 
review  
Contact: ande2445@umn.edu  
☐ Yes 
☒ No 
Include the use of 
recombinant or synthetic 
nucleic acids, toxins, or 
infectious agents? 
Complete the IBC application via 
eprotocol.umn.edu 
Contact: 
☐ Yes 
☒ No 
Include the use of human fetal 
tissue, human embryos, or 
embryonic stem cells? 
Contact OBAO for submission 
instructions and guidance 

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☒ Yes 
☐ No 
Include PHI or are you 
requesting a HIPAA waiver? 
If yes, HIPCO will conduct a review of 
this protocol. 
Contact: privacy@umn.edu  
☐ Yes 
☒ No 
Use data from the Information 
Exchange (IE)? 
The Information Exchange ancillary 
review will be assigned to your study by 
IRB staff 
Contact: ics@umn.edu  
Approval 
must be 
received 
prior to IRB 
approval. 
 
These 
groups do 
not have a 
separate 
application 
process but 
additional 
information 
from the 
study team 
may be 
required. 
 
☐ Yes 
☒ No 
Use the Biorepository and 
Laboratory Services to collect 
tissue for research? 
The BLS ancillary review will be 
assigned to your study by IRB staff. 
Contact: cdrifka@umn.edu  
☐ Yes 
☒ No 
Have a PI or study team 
member with a conflict of 
interest? 
The CoI ancillary review will be 
assigned to your study by IRB staff 
Contact: becca002@umn.edu  
☒ Yes 
☐ No 
Need to be registered on 
clinicaltrials.gov? 
If you select “No” in ETHOS, the 
clinicaltrials.gov ancillary review will be 
assigned to your study by IRB staff 
Contact: kmmccorm@umn.edu  
☐ Yes 
☒ No 
Require registration in 
OnCore? 
If you select “No” or “I Don’t Know” in 
ETHOS, the OnCore ancillary review will 
be assigned to your study by IRB staff 
Contact: oncore@umn.edu  
Does not 
affect IRB 
approval. 
 
 
 
 

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REVISION HISTORY 
 
Revision # 
Version Date 
Summary of Changes 
Consent 
Change? 
1 
3/25/2020 
We have made changes to clarify two points. 
First, we ensured that the compensation 
listed for all documents is $80. Second, we 
have clarified how the Physical Activity 
Readiness Questionnaire will be scored as it 
pertains to inclusion and exclusion (Sections 
8.1 and 13.1). Notably, we have also better 
clarified scoring in the phone-based 
screening questionnaire to be used by the 
research team to screen participants for 
inclusion in the trial. 
No. 
2 
4/14/2021 
We have updated this protocol to reflect:  
(1) the precautions our research team will be 
taking to reduce the potential for SARS-CoV-2 
transmission; (2) the methodology we will 
use to deliver this trial in a remote manner 
if/when in-person exercise sessions are not 
possible due to the COVID-19 pandemic 
and/or participant preference (Sections 4.1 
and 5.2). Additionally, the protocol was 
edited for clarity and to avoid redundancies. 
No. 
3 
05/25/2021 
We have updated this protocol to reflect: 
- The removal of waist circumference 
measures at the in-person visits 
- the removal of a fasting blood draw 
measure at week 4 
- the removal of a study questionnaire at 
each assessment visit 
Yes. See 
updates dated 
5-25-2021 
4 
07/21/2021 
We have updated this protocol to specifically 
add ResearchMatch as a recruitment 
methodology (See Section 11.1). We have 
also re-considered our compensation amount 
and have made a nominal increase in amount 
(See Section 11.4). 
Yes. See 
updates dates 
7-21-2021. 
5 
09/08/2021 
We removed the use of Proton-Pump 
Inhibitors as one of the exclusionary criteria 
(see section 8.2) 
 
 
 
 
 

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Revision # 
Version Date 
Summary of Changes 
Consent 
Change? 
6 
05/11/2022 
We have removed the upper limit for BMI as 
one of the inclusion criteria(see section 8.1 & 
8.2) 
 
 
 
 

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Table of Contents 
 
1.0 
Objectives ............................................................................................................. 8 
2.0 
Background........................................................................................................... 8 
3.0 
Study Endpoints/Events/Outcomes .................................................................... 11 
4.0 
Study Intervention(s)/Investigational Agent(s) .................................................... 12 
5.0 
Procedures Involved ........................................................................................... 13 
6.0 
Data and Specimen Banking ................................................................................ 18 
7.0 
Sharing of Results with Participants .................................................................... 19 
7.1 
Sharing Results: .................................................................................................. 19 
7.2 
Sharing Genetic Results: ..................................................................................... 19 
7.2.1 Disclosure of Results: ............................................................................................ 19 
8.0 
Study Population ................................................................................................ 20 
9.0 
Vulnerable Populations ....................................................................................... 22 
10.0 Local Number of Participants .............................................................................. 23 
11.0 Local Recruitment Methods ................................................................................ 24 
12.0 Withdrawal of Participants ................................................................................. 25 
13.0 Risks to Participants ............................................................................................ 25 
14.0 Potential Benefits to Participants ........................................................................ 26 
15.0 Statistical Considerations .................................................................................... 26 
16.0 Health Information and Privacy Compliance ....................................................... 28 
16.1 Select which of the following is applicable to your research: .............................. 28 
17.0 Confidentiality .................................................................................................... 32 
18.0 Provisions to Monitor the Data to Ensure the Safety of Participants ................... 32 
19.0 Provisions to Protect the Privacy Interests of Participants .................................. 33 
20.0 Compensation for Research-Related Injury ......................................................... 34 
21.0 Consent Process ................................................................................................. 34 
22.0 Setting ................................................................................................................ 35 
23.0 Multi-Site Research............................................................................................. 35 
24.0 Coordinating Center Research ............................................................................ 36 
25.0 Resources Available ............................................................................................ 36 
26.0 References .......................................................................................................... 37 
 

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ABBREVIATIONS/DEFINITIONS 
● CMD: cardiometabolic disease - envelops numerous disease states like 
prediabetes, diabetes, metabolic syndrome, and cardiovascular disease. 
● DNA: deoxyribonucleic acid - to be extracted from the stool samples 
provided by participants. 
● PreD: prediabetes - the cardiometabolic disease of interest in the current 
study, with the formal definition provided within text. 
● PA: physical activity - any bodily movement that raises energy expenditure 
above resting levels. 
● PCR amplified: polymerase chain reaction amplification - widely used 
process to generate information on the relative abundance of different 
microbial groups when analyzing the 16S rRNA obtained from stool samples. 
● rRNA: ribosomal ribonucleic acid 
● SCFA: short chain fatty acids - fatty acids with fewer than 6 carbon atoms 
that are generated in the gut and perform select physiological functions 
important to, among other things, gastrointestinal health. 
● T2D: type 2 diabetes - highly prevalent cardiometabolic disease that up to 
70% of prediabetics will convert to if steps are not taken to impede this 
conversion. 
● QIIME, DADA2, phyloseq, DESeq2, and SAS: software packages to be used in 
the proper analyses of all study data. 
 
 

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1.0 
Objectives 
1.1 Purpose: We propose a 100-participant randomized controlled 2-arm 
parallel trial which employs a metagenomic approach to examine how 8 
weeks of supervised moderate-intensity treadmill walking exercise 
(MWE)1,2 for 30-45 min 3 times/week alters the gut microbiome, serum 
SCFAs, and the cardiometabolic profile, body weight, and body composition 
of individuals 30-64 years old who are overweight or obese and have PreD. 
Aim 1: Compare the effect of 8 weeks of MWE vs. control on shifts in the 
gut microbiome of overweight or obese individuals with PreD. H1: Gut 
microbial diversity and functional capacity (specifically, genes relevant to 
SCFA production) will increase improve after 8 weeks in the experimental 
group vs. control. 
Aim 2: Compare the effect of 8 weeks of MWE vs. control on changes in 
serum SCFAs. H2: Serum Levels of SCFAs butyrate, acetate, and propionate 
will increase after 8 weeks in the experimental group vs. control.  
Aim 3: Use formal mediation methods to estimate the proportion of 
MWE-induced changes in participants’ cardiometabolic profiles, body 
weight, and body composition that are mediated by changes in the gut 
microbiome and/or serum SCFAs. H3: MWE-induced changes in the gut 
microbiome and serum SCFAs will partly explain changes in participants’ 
cardiometabolic profile (e.g., blood lipids, fasting insulin/glucose, blood 
pressure, waist circumference), body weight, and body composition. 
 
2.0 
Background 
2.1 Significance of Research Question/Purpose: In the U.S, 91.8 million adults 
have prediabetes (PreD).3 The prediabetic state is a prominent public 
health problem as up to 70% of individuals with PreD convert to type 2 
diabetes (T2D).4 The risk of cardiovascular disease (CVD) is high among 
those with PreD and T2D, and nearly $330 billion/year is spent treating 
T2D.3,5-7 The American Heart Association (AHA) and American Diabetes 
Association (ADA) have thus called for continued investigation into 
mechanisms connecting health behaviors to pre-clinical cardiometabolic 
disease (CMD; e.g., PreD) to reduce future disease risk.8,9 Physical activity 
(PA) is a key modifiable determinant of good health.10 Literature suggests 
PA recommendation10 adherence is related to a 25-35% dose-dependent 
reduction in all-cause mortality1,11,12, with as little as 75 min/week of 
regular moderate-intensity PA beneficial.1 Data from the Diabetes 
Prevention Program (DPP) suggested that, among those with PreD, a 
behavioral intervention emphasizing PA and diet to be at least as successful 
as metformin in preventing T2D, partially due to weight loss during this 

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trial.13 Yet, many individuals do not fully realize the cardiometabolic 
benefits of PA, and mechanistic pathways linking PA and cardiometabolic 
improvements are not fully understood.8,9,14,15 The gut microbiome16 has 
been posited as a mechanistic intermediate linking heath behaviors such as 
PA to CMD development.14,17-19  
The gut microbiome may be important for immunological,20,21 
metabolic,16,22 inflammatory,18,23,24 and neurobehavioral processes22,24,25, 
some of which might partially explain how health behaviors influence CMD 
risk. Yet, limited evidence exists characterizing the effect of PA on the 
human gut microbiome. To date, most studies of the impact of PA 
interventions on the gut microbiome have used animal models,26-41 with 
these studies supporting regular chronic PA (i.e., exercise) as a modulator 
of the gut microbiota. Animal studies have most often reported exercise-
related changes to the Firmicutes27-31,38 or Bacteroidetes26,34,35,40 phyla 
while also noting beneficial alterations to microbial community 
diversity26,29,30,32-35,38-40 and short chain fatty acid (SCFA)-producing taxa33-
36,38,41. SCFAs are reported to promote gastrointestinal health, increase 
energy expenditure42, and to be related to a lean phenotype.14,27,41 The five 
existing human gut microbiome trials43-47 most often implemented non-
controlled studies of 5- to 8-weeks of moderate-intensity exercise on 3 
days/week for 30-60 min/session. Among overweight or obese adults, prior 
studies43-45 note increased -diversity and altered ꞵ-diversity as a result of 
exercise; similar findings emerged in another study of healthy older 
adults.46 Additionally, one study noted exercise to result in increases in 
SCFAs (in stool), and corresponding changes to SCFA-related bacterial 
genes and taxa,45 while two other studies observed improvements in the 
metabolic potential of the gut microbiome43,44. These findings corroborate 
observational studies in humans wherein more physically active individuals 
have greater microbiota diversity and/or improved capacity to produce 
SCFAs than less active individuals.48-53 To our knowledge, no randomized 
controlled parallel design study has been conducted using metagenomic 
profiling (i.e., assessments of microbial functional capacity) to examine 
the influence of exercise on the human gut microbiome and serum SCFA 
levels in a population with a pre-clinical CMD. Moreover, no study has 
tested if changes in these outcomes mediate changes in cardiometabolic 
profiles or body weight and composition.  
The proposed study is innovative for the following reasons. First, we know 
of no other study in individuals who are overweight or obese with PreD 
that has examined how an exercise program may modify the gut 
microbiome and serum SCFAs. Second, we will study, for the first time, the 
interrelationship between exercise and the gut metagenome using a 
recently validated method for ‘shallow shotgun’ sequencing.54 The shallow 
shotgun approach enables interrogation of the gut metagenome at a cost 

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that scales to longitudinal repeated measures study designs. As such, we 
will generate novel data on longitudinal changes in the gut metagenome 
resulting from MWE in individuals with PreD via thorough examination of 
the function of various microbial taxa and their effect on health over 
time.55 Finally, the use of formal mediation analyses to examine the degree 
to which MWE-related changes in the preceding outcomes mediate 
changes in cardiometabolic risk profiles is novel and will inform the relative 
importance of the gut microbiome in exercise-related cardiometabolic 
improvements. These mediation analyses will also allow for a deeper 
interpretation of the physiological mechanisms by which exercise promotes 
improved health—addressing calls by the AHA and ADA.8,9. 
2.2 Preliminary Data: To date, only five human trials have investigated the 
effect of exercise on the gut microbiome.43-47, with none of these studies 
having been conducted in a population with a subclinical CMD (e.g., PreD). 
Further, there are notable limitations to these human studies. First, aside 
from Kern et al.43, no study employed a true control group. Second, most 
studies were conducted in small samples of apparently healthy adults, with 
only two studies44,47 incorporating a true metagenomic sequencing 
approach. Incorporating a metagenomic approach (i.e., assessing microbial 
genes and functional capacity) allows for a deeper understanding of 
microbial diversity and ecology as well as better elucidation of the function 
of various microbial taxa and their effect(s) on health.55 Finally, Allen et 
al.45 only assessed SCFAs changes in stool and not in serum. 
2.3 Existing Literature: The gut microbiome16 has been posited as a 
mechanistic intermediate linking heath behaviors such as PA to CMD 
development.14,17-19 The gut microbiome may be important for 
immunological,20,21 metabolic,16,22 inflammatory,18,23,24 and 
neurobehavioral processes22,24,25, some of which might partially explain 
how health behaviors influence CMD risk. Yet, limited evidence exists 
characterizing the effect of PA on the human gut microbiome. To date, 
most studies of the impact of PA interventions on the gut microbiome have 
used animal models,26-41 with these studies supporting regular chronic PA 
(i.e., exercise) as a modulator of the gut microbiota. Animal studies have 
most often reported exercise-related changes to the Firmicutes27-31,38 or 
Bacteroidetes26,34,35,40 phyla while also noting beneficial alterations to 
microbial community diversity26,29,30,32-35,38-40 and short chain fatty acid 
(SCFA)-producing taxa33-36,38,41. SCFAs are reported to promote 
gastrointestinal health, increase energy expenditure42, and to be related to 
a lean phenotype.14,27,41 The five existing human gut microbiome trials43-47 
most often implemented non-controlled studies of 5- to 8-weeks of 
moderate-intensity exercise on 3 days/week for 30-60 min/session. Among 
overweight or obese adults, prior studies43-45 note increased -diversity and 
altered ꞵ-diversity as a result of exercise; similar findings emerged in 

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another study of healthy older adults.46 Additionally, one study noted 
exercise to result in increases in SCFAs (in stool), and corresponding 
changes to SCFA-related bacterial genes and taxa,45 while two other studies 
observed improvements in the metabolic potential of the gut 
microbiome43,44. These findings corroborate observational studies in 
humans wherein more physically active individuals have greater microbiota 
diversity and/or improved capacity to produce SCFAs than less active 
individuals.48-53 
 
3.0 
Study Endpoints/Events/Outcomes 
3.1 Primary Endpoint/Event/Outcome: Expanded data collection procedures 
for our primary and secondary outcomes are available in Section 5.2. We 
have two Primary Outcomes. The first primary outcome is changes to the 
gut microbiome, assessed via metagenomic sequencing, from pre- to post-
intervention (Specific Aim 1). The Shannon Index—a measure of microbial 
community diversity with good reproducibility (ICCs>0.50)56—will be our 
primary indicator for changes in gut microbiota composition, and will be 
assessed at baseline, and following the 4th and 8th weeks. The second 
primary outcome is serum SCFA levels. Participants will undergo fasted 
blood draws to assess serum SCFA levels (Specific Aim 2). This work will be 
performed using the Metabolon pipeline.57  
3.2 Secondary Endpoint(s)/Event(s)/Outcome(s): We have several Secondary 
Outcomes. For Specific Aim 3 cardiometabolic profiles will be assessed via 
assays for high-density lipoprotein cholesterol (HDL-C), triglycerides, fasting 
insulin/glucose, and c-reactive protein (CRP). All blood assays will be 
conducted by the UMN Advanced Research and Diagnostic Laboratory (see 
‘Facilities’ document). Blood samples collected will be stored at -80C prior 
to batch-processing. Resting blood pressure (BP) will be taken using an 
Omron Automatic BP Cuff (Omron; Lake Forest, IL), with the average of the 
last two BP measurements reported. These cardiometabolic indices form 
the foundation of the ‘metabolic syndrome’,59-63 and we will form a 
composite continuous score for cardiometabolic disease risk from HDL-C, 
fasting glucose, triglycerides, the average of systolic and diastolic BP, and 
waist circumference. Specifically, z-scores for each biomarker will be 
calculated and the within-participant average across z-scores computed 
and referred to as cardiometabolic disease risk (CMDrisk; the primary 
dependent variable for Specific Aim 3). This score has been used in 
epidemiologic, experimental, and clinical studies.62,64-67 Individual 
components will be described in secondary analyses as needed. We will 
also measure HbA1c using whole blood analyzed at the UMN Advanced 
Research Diagnostic Laboratory;  the A1cNOW+® device may also be used 
in a limited capacity to assess validity.68 The oral microbiome will be 

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assessed using a widely employed salivary testing technique. Briefly, we 
will ask that participants chew a paraffin tablet (Orion Diagnostica) for 5 
minutes after which the participant will be asked to spit into a 50mL tube 
until 5mL of saliva is collected. We have experience with this non-invasive 
assessment69, with the assessment taking no longer than 5-10 minutes 
overall and presenting no greater than minimal risk. Other secondary 
outcomes are necessary to measure to ensure we can properly control for 
confounding. Dietary composition will be assessed via 3 phone-based 
dietary recalls during the run-in phase, and again during the final two 
weeks of the study. We will use these dietary recalls collected during the 
run-in period to develop personalized 3-day meal plan to standardize 
individual dietary behavior prior to stool sample collection. Employing 
dietary recalls at these time points will also allow us to adequately 
statistically control for participants’ dietary changes—changes which, if not 
controlled, might confound study results.70,71 Physical activity will be 
measured for the duration of the study using the Fitbit Inspire 2 
smartwatch that uses accelerometry to measure physical activity metrics 
such intensity, steps, etc. Finally, anthropometric outcomes will include 
height, weight, and body composition. These measures, along with the 
other secondary outcomes aside from PA and Dietary composition, will be 
measured at the end of the 3-week run-in phase, and at the end of the 4th 
and 8th study weeks.  
4.0 
Study Intervention(s)/Investigational Agent(s) 
4.1 Description: Following baseline data collection, participants will be 
randomized into one of two study groups using PROC PLAN (SAS 9.4) and a 
1:1 allocation ratio.  
GROUP 1 (aka, Experimental group): participants will complete 3 walking 
sessions/week  for a total of 8 weeks (24 total sessions). Walking sessions 
will either take place on new commercial treadmills in the Epidemiology 
Clinical Research Center or remotely at or around a participant’s home (see 
below). Regardless of the walking location, walking sessions will be 30 min 
in duration during intervention weeks 1 through 4 and a minimum of 45 
min each during intervention weeks 5 through 8.  
GROUP 2 (aka, Control group): participants will be asked to maintain their 
usual PA during the initial 8-week intervention period but will undergo all 
data collection procedures as outlined for the experimental group (See 
Section 5.2).  
 
Experimental group participants who want to forego in-person exercise 
sessions on the University of Minnesota campus due to logistical 
constraints or the COVID-19 pandemic will modify the exercise 
intervention in the following manner:  

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Experimental group participants will engage in the same frequency and 
duration of walks as described above, but with a change in location to 
either: (a) in neighborhood or backyard walk (weather permitting, 
maintaining physical distancing); (b) on a treadmill within their home; and 
(c) in the hallway of their home if options a. and b. are not possible. 
 
To monitor physical activity levels and ensure remote compliance: we will 
monitor to ensure that participants are completing their home-based walks 
at the proper duration and intensity using the Fitabase® platform. (See 
more information on the data security of Fitabase® in Section 16.7)                                            
4.2 Drug/Device Handling: N/A 
 
4.3 Biosafety: N/A 
 
4.4 Stem Cells: N/A 
 
5.0 
Procedures Involved 
5.1 Study Design: We will employ a 100-participant, 8-wk, randomized 2-arm 
parallel trial design with a 1:1 allocation ratio. 
5.2 Study Procedures: All eligible and consented participants will complete a 3-
week run-in phase during which baseline outcome assessments will be 
performed. During run-in weeks 1 and 2, participants will wear a Fitbit 
Inspire 2 smartwatch and complete three unannounced dietary recalls. 
During the run-in week 3 (see table below), participants will consume their 
3-day standardized meal plan on days three through five, obtain a fecal 
sample, and complete a study assessment visit. To quantify compliance 
with the 3-day meal plan, participants will be provided with a paper 
checklist of all foods to be consumed, and will be asked to indicate which 
foods were consumed and to document any deviation. The study 
assessment visit will include a fasting blood draw, blood pressure in 
triplicate, weight measure and body fat assessment in duplicate, and a 
saliva sample. Following successful completion of all measures, participants 
will then be randomized into one of the two study groups using PROC PLAN 
(SAS 9.4) and a 1:1 allocation ratio. 
 
Group 1 (Experimental group): Participants will complete 3 treadmill 
walking sessions/week (24 total sessions) over 8 weeks at or around their 
home or in-person within the Epidemiology Clinical Research Center. Each 
walking sessions will be 30 min in duration during intervention weeks 1 

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through 4 and 45 min during intervention weeks 5 through 8. Notably, this 
exercise dose (i.e., intensity x session duration x weekly frequency x 
intervention length) is equivalent to, or greater than, that employed in 
most existing human studies of exercise and the gut microbiome in 
addition to the exercise dosage observed necessary to yield meaningful 
cardiometabolic changes.72 Therefore, while the dose is ‘modest’, it reflects 
what is relevant in real-world interventions and appropriate for overweight 
or obese sedentary individuals.  
 
During week 4, participants will again consume their 3-day standardized 
meal plan, obtain a fecal sample, and complete a study assessment visit 
(same measures as described above for the 3-week run-in, except no 
fasting blood draw will be taken).  
 
During weeks 7 and 8, participants will complete 3 unannounced phone-
based dietary recalls. During week 8, participants will again consume their 
3-day standardized meal plan, obtain a fecal sample, and complete a study 
assessment visit (same measures as described above in run-in week 3).  
Table 1. Timing of Data Collection events during the 3-Week Run-In and 
Intervention Weeks 4 & 7/8. 
Day 1 
Day 2 
Day 3 
Day 4 
Day 5 
Day 6 
Day 7 
 
 
 
 
Stool 
(pm)* 
Stool 
(am)* 
Stool 
 
 
Standard 
Diet 
Day1 
Standard 
Diet 
Day2 
Standard 
Diet 
Day3 
 
 
 
 
 
 
 
 
Assess. 
Visit 
*Preferred timing stool collection is the evening of Day 5 or morning of Day 6, though 
anytime in the Day 5 – 7 window is acceptable.  
  
Group 2 (Control group): Participants will be asked to maintain their usual 
PA during the initial 8-week intervention period but will undergo all data 
collection procedures as outlined above for the experimental group.  
 
Expanded Data Collection Procedures: We have two Primary Outcomes. 
The first primary outcome is changes to the gut microbiome, assessed via 
metagenomic sequencing, from pre- to post-intervention (Specific Aim 1). 
The Shannon Index will be our primary indicator for changes in gut 
microbiota composition. Participants will use the OMNIgene-Gut stool 
collection kit to obtain their fecal samples collected at baseline, week 4 and 

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week 8. The home-collected samples will be returned to our lab within 30 
days. DNA will be extracted from the stool samples using the 96 well plate 
Mo Bio Power soil kit on an Epmotion robot and eluted into 50ul aliquots. 
Each extraction will contain negative and positive controls representing 
defined bacterial communities (Zymo; Irvine, CA). DNA will be quantified 
using Quant-it and stored at -80°C. Metagenomic libraries will be prepared 
at the UMN Genome Center (see ‘Facilities’ document) using the Nextera 
XT kit (Illumina; San Diego, CA) and barcoded. DNA libraries will be 
denatured with NaOH and diluted to 8-12 pM in Illumina’s HT1 buffer, 
spiked with 1% PhiX and a HiSeq 1x100 cycle v3 ki Illumina HiSeq, with an 
expected output of at least 0.5 million total sequences per sample. This 
approach has been shown to be valid54 and represents an approach that 
can address both gut microbial taxonomy and community functional 
content at a cost similar to 16S studies.  
The second primary outcome is serum SCFA levels. Participants will 
undergo fasted blood draws to assess serum SCFA levels (Specific Aim 2). 
This work will be performed using the Metabolon pipeline.57 Briefly, serum 
samples are spiked with a solution of eight stable labelled internal 
standards and subjected to protein precipitation. After centrifugation, an 
aliquot of the supernatant is derivatized. The reaction mixture is analyzed 
by LC MS/MS on an Agilent 1290/ AB Sciex 5500 system. Peak areas of the 
respective analyte product ions are measured against the peak area of the 
corresponding internal standard product ions. Quantitation is performed 
using a weighted least squares regression analysis generated from fortified 
calibration standards prepared immediately prior to each run.  
We have several Secondary Outcomes. For Specific Aim 3 cardiometabolic 
profiles will be assessed via assays for high-density lipoprotein cholesterol 
(HDL-C), triglycerides, fasting insulin/glucose, and c-reactive protein (CRP). 
All blood assays will be conducted by the UMN Advanced Research and 
Diagnostic Laboratory (see ‘Facilities’ document). Blood samples collected 
will be stored at -80C prior to batch-processing. Resting blood pressure 
(BP) will be taken using an Omron Automatic BP Cuff (Omron; Lake Forest, 
IL), with the average of the last two BP measurements reported. These 
cardiometabolic indices form the foundation of the ‘metabolic 
syndrome’,59-63 and we will form a composite continuous score for 
cardiometabolic disease risk from HDL-C, fasting glucose, triglycerides, the 
average of systolic and diastolic BP. Specifically, z-scores for each 
biomarker will be calculated and the within-participant average across z-
scores computed and referred to as cardiometabolic disease risk (CMDrisk; 
the primary dependent variable for Specific Aim 3). This score has been 
used in epidemiologic, experimental, and clinical studies.62,64-67 Individual 
components will be described in secondary analyses as needed. We will 
also measure HbA1c using standardized procedures and the blood 

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collected. The oral microbiome will be assessed using a widely employed 
salivary testing technique. Participants will be asked to drool into a 50mL 
tube until 5mL of saliva is collected.  For participants that are having 
difficulty generating saliva, we will ask those participants chew a small 
piece of orthodontic wax for 5 minutes and then drool into the 50mL tube. 
We have experience with this non-invasive assessment69, with the 
assessment taking no longer than 5-10 minutes overall and presenting no 
greater than minimal risk. Other secondary outcomes are necessary to 
measure to ensure we can properly control for confounding. Dietary 
composition will be assessed via 3 phone-based dietary recalls. These 
recalls will be performed on 2 weekdays and 1 weekend day by trained 
interviewers. Assessment of diet on multiple days allows us to capture the 
individual variation in participants’ diets.71 We will use these dietary recalls 
to develop personalized 3-day meal plan to standardize individual dietary 
behavior prior to stool sample collection. Although randomization should 
lead to approximately balanced groups with respect to diet, employing 
dietary recalls at these time points will allow us to adequately statistically 
control for participants’ dietary changes—changes which, if not controlled, 
might confound study results.70,71 Physical activity will be measured during 
the duration of the study using the Fitbit Inspire 2 smartwatch. Finally, 
anthropometric outcomes will include height measured using a Seca 
stadiometer (Seca; Hamburg, Germany), with weight and body composition 
measured using the Tanita TBF-300a Body Composition Analyzer (Tokyo, 
Japan).  
To collect blood samples and saliva samples as well as body weight, body 
fat percentage, and blood pressure, we are proposing to employ the 
following procedures in the designated settings while following all 
necessary COVID-19 precautions: 
Setting: Baseline, week 4, and week 8 visits will occur in the Epidemiology 
Clinical Research Center (ECRC). We will follow all UMN, OVPR and ECRC 
approved guidelines for conducting research during the COVID-19 
pandemic.  
COVID-19 Screening: Participants will complete the OVPR-approved ECRC 
COVID-19 phone screener the day prior to their visit. Prior to entering the 
ECRC, participants will call the study coordinator and complete the 
screening questionnaire again. Upon entry into the building, study staff will 
assess the participant’s temperature.  
• Personal Protective Equipment (PPE): Participants will be asked to 
wear a mask. All ECRC staff coming within 6 feet of study 
participants (e.g. for blood or blood pressure collection) will be 
adhering to UMN guidelines for PPE (e.g. masks, gloves). 

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ECRC Data Collection: 
• Each participant will be directed to a well-ventilated exam room, 
outfitted with a HEPA air purifier for additional protection where all 
non-lab measurements will take place.  
• Participant will step upon the study scale and state to us their 
weight and body fat percentage while we stand at least 6 feet away. 
No other participants will be in the exam room, so confidentiality 
will be assured.  
• Study staff will obtain blood pressure and a trained phlebotomist 
will collect the blood samples. As always, juice and snacks will be on 
hand in case needed after the blood draws. We will then have 
participants collect their saliva samples. 
• Participant will be directed out of the building. Blood samples will 
be processed immediately and placed into a -80 freezer. 
• We will thoroughly disinfect all touchpoints and research materials 
while properly disposing of biohazards. 
If ECRC visits cannot occur due to continued COVID-19 outbreaks, we will 
conduct Home-Based Visits in the following manner: 
• Participants will complete the OVPR-approved ECRC COVID-19 
phone screener the day prior to their visit. 
• Upon staff arrival, study staff will call the participant to again 
complete the OVPR-approved ECRC COVID-19 phone screener, and 
a temperature reading of the participant will be taken when they 
come outside. 
• The scale platform will be placed outside the participant’s home on 
a flat surface. The scale reader is connected to the platform by a 6 
foot cord, enabling the staff to read the output while maintaining 
physical distance.   
• Blood pressure will be obtained by the study staff member 
completing the research session. 
• The study phlebotomist will collect the blood samples with 
participant seated in the chair. Juice and snacks will be made 
available in case the need arises. Blood will be placed in a cooler 
with dry ice for transport back to the ECRC for processing and 
placement into a -80 freezer. 
o We will also thoroughly disinfect all touchpoints and 
research materials while properly disposing of biohazards. 
 
We have outlined the procedures we will take to ensure participant safety 
during the intervention within section 13.1. This study presents no greater 
than minimal risk to participants. All data collection forms are available on 
ETHOS.  

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5.3 Study Duration: The total duration of study participation will be 
approximately 11 weeks from initial contact with the researchers to the 
conclusion of all study procedures.  
 
As for the data analyses associated with this study, we anticipate these 
data analyses to take approximately 4 months—allowing time for the UMN 
Genomics Center and the UMN Advanced Research and Diagnostic 
Laboratory to batch process participants’ stool samples and blood work, 
respectively. 
 
5.4 Individually Identifiable Health Information: Each participant will be 
assigned a unique study ID that will consist of a series of numbers and 
letters that will have no meaning to any of the individual's personal 
identifying characteristics. The investigators will use this ID to track each 
individual participant in the research database. There will be not identifiers 
in this database. The identifiers will be maintained in separate, secure, 
encrypted, password-protected database in a different physical location. 
This separate database for the identifying information will also include the 
study ID so that the investigators can link research data to individuals when 
necessary. 
 
5.5 Use of radiation: N/A 
 
5.6 Use of Center for Magnetic Resonance Research: N/A 
 
6.0 
Data and Specimen Banking 
6.1 Storage and Access: We will initially collect data on paper after which we 
will transfer these data to computer spreadsheets for export to statistical 
packages, with this data entry conducted separately by two individuals to 
ensure data integrity. All PHI will be stored in a folder on the University of 
Minnesota’s Box drive which is HIPAA compliant. All paper-based data will 
be stored in a locked file cabinet within the West Bank Office Building near 
the PI’s office for a minimum of three years after the last participant has 
completed data collection. We will also store the de-identified electronic 
data for a minimum of three years on a server within the Division of 
Epidemiology and Community Health.  
Buffy coat and serum from blood draws and saliva from salivary collection 
will be stored for up to 20 years in the PI’s freezers located at the 

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Epidemiology Clinical Research Center or MoosT 1-355. All NIH procedures 
and University policies for the protection of confidentiality will be followed. 
Identifying data will be kept confidential, and only de-identified data will be 
shared. 
 
6.2 Data: We will collect the following data: Serum, plasma, buffy coat, saliva, 
stool samples, Fitabase activity app data, food recall data, questionnaire 
data, and anthropometric data. The following data will be banked for 
future use if the participant consents: Serum, plasma, buffy coat, stool, and 
saliva. Otherwise, we will properly dispose of these specimens. All data will 
only be linked via a unique identification number. No identifying 
information will be included. 
 
6.3 Release/Sharing: Materials generated under the project will be 
disseminated in accordance with University and NIH policies. Depending on 
such policies, materials may be transferred to others under the terms of a 
material transfer agreement. Access to databases and associated software 
tools generated under the project will be available for educational, 
research, and non-profit purposes. Such access will be provided using web-
based applications, as appropriate. Publication of data shall occur during 
the project, if appropriate, or at the end of the project, consistent with 
normal scientific practices. Research data which documents, supports and 
validates research findings will be made available after the main findings 
from the final research data set have been accepted for publication. Such 
research data will be redacted to prevent the disclosure of personal 
identifiers. 
 
7.0 
Sharing of Results with Participants 
7.1 Sharing Results: Following the conclusion of the study, we will share with 
each participant their anthropometric (weight, waist circumference) and 
cardiometabolic data (fasting insulin/glucose, triglycerides, cholesterol 
levels, and blood pressure) collected during the 2-week run-in 
visit/baseline fasting visit and following the 4th and 8th weeks. We will share 
these data in a personalized letter to each participant. Further, we will 
provide participants with an overall aggregated summary of the study’s 
observations and/or any published manuscripts. 
 
7.2 Sharing Genetic Results: N/A 
 
7.2.1 Disclosure of Results: N/A 
 

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7.2.2 Returning Results to Participants: N/A 
 
Aggregate or individual results: N/A 
 
Laboratory results: N/A  
Plan for return of results to participants: N/A 
 
Types of results to be returned to participants: N/A 
 
7.2.3 Future analysis of genotypes: N/A 
 
 
8.0 
Study Population 
8.1 Inclusion Criteria:  
(1) 30-64 years old.  
(2) Classified as overweight or obese with BMI >25.0kg/m2, no upper limit 
(3) Documentation* of a PreD diagnosis within one year of enrollment by 
physician or primary care provider based on lab tests showing a fasted 
blood glucose of 100-125 mg/dL, a 2-hour oral glucose tolerance test of 
140-199 mg/dL, or an HbA1c level of 5.7%-6.4%76; OR a study screening 
lab value of HbA1C within the afore mentioned range.  
(4) Currently engaged in <100 min/week of PA—confirmed via the 
Modifiable Activity Questionnaire.77 
(5) No exercise contraindications as assessed by the Physical Activity 
Readiness Questionnaire (PAR-Q)78—this Questionnaire involves seven 
“yes” or “no” questions regarding an individual’s health status, with 
answering “yes” to any one of these questions requiring a prospective 
participant to acquire a written doctor’s note stating they can safely 
participate in the trial’s exercise intervention. They would not be 
enrolled until this doctor’s note is received. 
(6) No self-reported physical/mental disabilities or gastrointestinal 
conditions. 
(7) No antibiotic usage within the last 45 days.  
(8) Stable weight over the last 6 months (<10% change). 
(9) Not currently pregnant, planning to become pregnant, or currently 
breastfeeding. 
(10) Willing to maintain current dietary and exercise habits, aside from any 
changes to be made per the study exercise protocol.  
 
*Note: Documentation can include either a print out or screen shot of 
the lab value illustrating eligibility, along with the date of the test and 
the participant’s name. If a hard copy is provided, the date and name 

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will be redacted.  
 
8.2 Exclusion Criteria:  
(1) Individuals <30 or >64: younger individuals may have not yet reached 
physiological maturity and in whom PreD prevalence is low; older 
individuals are more likely to have contraindications to PA and other 
comorbidities.  
(2) BMI< 25kg/m2. No Upper limit. 
(3) Individuals with an HbA1c level <5.7% or >6.4%. 
(4) Currently engaged in ≥100 min/wk of PA. 
(5) Individuals with contraindications to exercise participation as indicated 
by the PAR-Q. 
(6) A self-reported physical/mental disability that would prevent them from 
being able to adhere to the intervention. 
(7) A self-reported a gastrointestinal illness or condition. 
(8) Antibiotic use within the last 45 days.79-81  
(9) Self-reported use of metformin and/or other medications that could 
interfere with the primary outcome. 
(10) Unstable weight over the last six months (> 10% change). 
(11) History of bariatric surgery or a history of other medical interventions 
that would interfere with the primary outcome. 
(12) Currently pregnant or planning to become pregnant during the study 
period. 
(13) Currently breastfeeding. 
(14) Unwilling to be randomized to a study group. 
 
8.3 Screening: Interested individuals will be asked to complete a red-cap 
screening questionnaire (sent via email or with study staff over the phone) 
which will enable staff to pre-review possible participants for eligibility. 
Everyone who completes a screener will be contacted, and, if needed, 
screening responses can be clarified.  
 
Those who meet all eligibility criteria can then be scheduled for their 
consent/first study visit.  
 
Those who appear eligible but need to confirm that their HbA1c is within 
eligibility range will be scheduled to complete a consent/screening visit. At 
this visit, following completion of the consent/HIPAA form, participants will 
have their HbA1c tested. To determine their current HbA1c value, we will 
either use the A1cNOW+® device or collect a 2mL whole blood sample via 
venipuncture. 68 Of note, one of these methods will only be employed only 
after the participant has consented to be screened for the study. Briefly, 

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the A1cNOW+® device will use a small drop of blood obtained from a finger 
of a prospective participant to determine the individual’s HbA1C levels. 
This device has been validated against clinical laboratory measurements of 
HbA1C and is certified by the National Glycohemoglobin Standardization 
Program for this purpose.85 If a venipuncture sample is collected, this 
sample will be brought to the Advanced Diagnostic Research Laboratory for 
analysis. 
 
9.0 
Vulnerable Populations 
9.1 Vulnerable Populations:  
Population / Group 
Identify whether any of the 
following populations will be 
targeted, included (not necessarily 
targeted) or excluded from 
participation in the study.  
Children 
Excluded from Participation 
Pregnant women/fetuses/neonates 
Excluded from Participation 
Prisoners 
Excluded from Participation 
Adults lacking capacity to consent 
and/or adults with diminished 
capacity to consent, including, but 
not limited to, those with acute 
medical conditions, psychiatric 
disorders, neurologic disorders, 
developmental disorders, and 
behavioral disorders 
Excluded from Participation 
Non-English speakers 
Excluded from Participation 
Those unable to read (illiterate) 
Excluded from Participation 
Employees of the researcher 
Excluded from Participation 
Students of the researcher 
Excluded from Participation 
Undervalued or disenfranchised 
social group 
Excluded from Participation 

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Active members of the military 
(service members), DoD personnel 
(including civilian employees) 
Excluded from Participation 
Individual or group that is 
approached for participation in 
research during a stressful situation 
such as emergency room setting, 
childbirth (labor), etc. 
Excluded from Participation 
Individual or group that is 
disadvantaged in the distribution of 
social goods and services such as 
income, housing, or healthcare. 
Excluded from Participation 
Individual or group with a serious 
health condition for which there are 
no satisfactory standard treatments. 
Excluded from Participation 
Individual or group with a fear of 
negative consequences for not 
participating in the research (e.g. 
institutionalization, deportation, 
disclosure of stigmatizing behavior). 
Excluded from Participation 
Any other circumstance/dynamic 
that could increase vulnerability to 
coercion or exploitation that might 
influence consent to research or 
decision to continue in research. 
Excluded from Participation 
 
9.2 Additional Safeguards: N/A 
 
10.0 Local Number of Participants 
10.1 Local Number of Participants to be Consented: We will recruit participants 
on a rolling basis with the help of our clinical partners within community-
based health clinics and community outreach around Minneapolis-St. Paul. 
We will assume a 20% attrition rate based on documented attrition rates of 
PA interventions,82-84 along with a 30% rate of individuals who fail to fall 
within the HbA1c eligibility range during screening.  With these rates in 
mind, we will recruit 150 individuals total to meet our desired overall N of 
100 participants (n = 50 per group). Given the need for participants to be 

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able to participant in the exercise intervention at the Epidemiology Clinical 
Research Center, all participants will be recruited locally. 
 
11.0 Local Recruitment Methods 
11.1 Recruitment Process: Participants will be recruited on a rolling basis 
through a combination of the following methods: clinical referrals via 
partners within community-based health clinics, mass emails via University 
of Minnesota-Twin Cities campus listservs (all permissions will be obtained 
prior to this method), emails to past study participants who gave 
permission for future contact, and fliers posted on physical (e.g. campus 
buildings, community billboards) and virtual (e.g. Facebook, NextDoor) 
walls. Our study will also be searchable through clinicaltrials.gov and 
studyfinder.umn.edu. 
 
Should additional recruitment methods be needed, we may also use 
established recruitment databases and EMR obtained through partial 
HIPAA waiver (which we have had approved). For instance, we may use the 
patient access database overseen by the Clinical and Translational Science 
Institute.  
This study will also be listed at ResearchMatch.org. ResearchMatch is an 
electronic volunteer recruitment registry that allows people from 
anywhere in the country to self-register and express an interest in being 
prospectively considered for participation in research studies. This registry 
provides information about those volunteers to researchers who are 
looking for people to participate in studies, while protecting the privacy of 
the volunteers. 
 
We intend to begin this recruitment process in early 2021, and we believe 
we can recruit the requisite number of participants over one year. 
11.2 Identification of Potential Participants: Participants will be referred by one 
of our partnering care providers or will self-identify in response to fliers, 
emails, or internet postings.  
• 
Potential participants will make the initial contact with study staff by 
telephone, mail, text, or via completion of our online screening 
survey. Only after this initial contact will study staff reach back out to 
potential participants. 
 
11.3 Recruitment Materials: All study materials (fliers, postcards, emails) will 
contain a brief description of the study, primary inclusion criteria, a link to 
our study screening survey, and our contact information. Clinical 

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recruitment partners will receive materials in their preferred method, such 
as a small study postcard for placement in their lobby, or the same flier 
that we intend to post within community-based spaces such as local 
community centers. Emails sent will include the community flier as an 
attachment, while also containing similar text within the body of the email, 
along with a direct link to our study screening survey.  
 
11.4 Payment: Participants will have the choice of one of two incentives for 
study completion with greater than 80% adherence: (1) they may request 
$100 following their completion of the entire study, with this payment 
provided using the Greenphire ClinCard; OR (2) they may request to keep 
the Fitbit Inspire 2 along with a $25 ClinCard. This amount is reasonable 
given the study’s protocol and procedures. 
 
12.0 Withdrawal of Participants 
12.1 Withdrawal Circumstances: We do not foresee any circumstances under 
which participants will be withdrawn from the research without their 
consent. Participants who discontinue from the active intervention due to 
illness or injury will still be invited to complete all study measures at all 
data collection time points.  
12.2 Withdrawal Procedures: For participants who ask to withdraw from the 
study voluntarily, no additional data will be collected and participants 
would be compensated at the prorated rate as described in Section 11.4. 
would still be compensated according to the table above. However, data 
from the participant’s time in the study will still be used in data analysis 
when employing an intent-to-treat analysis approach. 
12.3 Termination Procedures: We do not foresee any circumstances that would 
result in the termination of the study for any individual. 
 
 
13.0 Risks to Participants 
13.1 Foreseeable Risks: The current study present no greater than minimal risk 
to participants. However, we will be taking precautions to ensure any 
potential risk from study participation is minimized. Per standard 
procedures during exercise intervention studies, we will ensure 
participants have no contraindications to exercise participation. However, 
we must note that any type of physical activity participation, even a 
walking exercise intervention like that of the current study, does carry risk 
in any population. Risks associated with exercise participation include 

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dizziness, nausea, muscle soreness, fatigue, fainting, joint pain, shortness 
of breath, muscular strains/sprains, and cramping. We will minimize these 
risks by allowing participants to complete a 5-10-minute warm-up of slow 
walking prior to each exercise session.  
 
Some potential psychological stress or embarrassment may also be 
associated with filling out the study questionnaire and/or undergoing 
measurements of weight, waist circumference, and blood pressure in 
addition to the collection of stool samples and saliva. Risks will be 
minimized by collecting this data in a private setting, either at their home 
or at the ECRC, depending upon the measure in question.  
 
As with all blood draws, there may be a modest amount of pain (equivalent 
to that of a pin prick) and lightheadedness associated with the collection of 
approximately 3 tablespoons of blood for each of three blood draws. We 
will minimize this risk by employing an experienced and trained 
phlebotomist for blood draws. Overall, the anticipated benefits to science 
outweigh the risks. 
 
Finally, to prevent any threat of a COVID-19 infection, we will follow all 
UMN/OVPR/ECRC protocols while the participant is at the ECRC and 
encourage masking and physical distancing if any walks are done outside 
of the ECRC environment.   
 
13.2 Reproduction Risks: N/A 
 
13.3 Risks to Others: N/A 
 
14.0 Potential Benefits to Participants 
14.1 Potential Benefits: Throughout the course of the 8-week intervention, 
participants may improve cardiometabolic indices (e.g., blood lipids, fasting 
insulin/glucose, blood pressure, c-reactive protein) while possibly 
experiencing weight loss too. 
 
15.0 Statistical Considerations 
15.1 Data Analysis Plan: Chi-square and independent t-tests will evaluate 
baseline group differences as appropriate. Mediation analyses will also be 
conducted. We outline our analysis plans in Section 15.3. 86-92 
 

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15.2 Power Analysis: Power for Aim 1 is based on analyses using alpha-diversity 
(Shannon Index) as the primary outcome. Taniguchi et al.46, reported a 
meanSD baseline Shannon Index of 5.70.6 which is consistent with 
preliminary data from gut samples collected by Dr. Demmer in other recent 
projects. Given this variance, and with a balanced design of n=50 in the 
experimental and control arms (N=100 total), we will have >99% power to 
detect a 1 unit increase in Shannon Index. Power for Aim 2 is based on 
changes in serum SCFAs. Our SCFA assays will be performed at Metabolon 
and our colleagues at Metabolon provided information on meanSD for 
these outcomes in their lab. They are as follows: acetate=18062500 
ng/mL, butyrate=3543 ng/mL, and propionate=5052 ng/mL. With these 
distributions, we will have >80% power to detect a difference in SCFA levels 
between treatment and control groups with a magnitude of at least 50% of 
1 SD.  
The primary outcome for Aim 3 will be a composite score based on the 
average of the z-scores for the following 5 biomarkers: HDL-C, fasting 
glucose, triglycerides, average of systolic and diastolic BP, and waist 
circumference. The standardized meanSD of this CMDrisk z-score in Dr. 
Pereira’s sedentary behavior intervention among 575 participants (Grant #: 
R01CA198971) is 00.7. This provides 80% power to detect a difference in 
the CMDrisk z-score of 0.4, between intervention and control at the 
conclusion of the study. 
 
15.3 Statistical Analysis: Aim 1 analysis: Gut microbiome data, derived via 
metagenomic sequencing, will be operationalized as described above. In a 
primary analysis, generalized linear regression models will regress Shannon 
Index on intervention assignment. The model is formalized as: E[Y]= 
intercept + β1*A + β2*C. E[Y]=expected Shannon value; β1=coefficient for 
intervention arm; β2=coefficient for a vector of confounders should this be 
necessary in our randomized design. Secondary analyses will consider 
metagenomic features (genes and functional profiles). Any metagenomic 
features not present at >0.1% relative abundance in at least 10% of 
samples will be removed. Zero-inflated log-linear generalized linear models 
with a Gaussian error term will be fitted, with treatment status as 
predictor, as implemented by the metagenomeSeq Bioconductor package 
for shotgun metagenomic data analyses.93  The generalized model, per 
metagenomic outcome, is formalized as: log E[Y]= intercept + β1*A + β2*C. 
E[Y]=expected normalized sequence count value; β1=coefficient for 
intervention arm; β2=coefficient corresponding to the vector of baseline 
confounders. Total read count is incorporated as an offset term. This 
analysis will generate a “top list” of genes, gene families, and pathways 
with altered abundance by intervention assignment. Separate models will 

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be developed for intervention status predicting week 4 and week 8 
outcomes. We will also consider time*treatment status models to 
incorporate stool sampling at all three timepoints. P-values will be 
corrected for multiple hypothesis testing using the false-discovery rate as 
we have done previously with high dimensional data.86-92,94-98  
Aim 2 analysis: As with Aim 1, generalized linear regression models will be 
used. Each of three SCFAs (acetate, butyrate and propionate) will be 
regressed on intervention assignment. The model is formalized as: E[Y]= 
intercept + β1*A + β2*C. E[Y]=expected SCFA value; β1=coefficient for 
intervention arm; β2=coefficient for a vector of confounders.  
Aim 3 analysis: We will use PROC CAUSALMED (SAS 9.4) to explore 
mediation effects on CMDrisk. This regression-based model was developed 
by Valeri and VanderWeele99 and VanderWeele.100 The predictor variable 
will be treatment (experimental vs. control) and the outcome will be 
CMDrisk. We will include age, sex as a biological variable, race/ethnicity, 
and PA duration as covariates. Mediation will be tested by adding 
parameters, in separate models, for gut microbiota diversity (8-week 
change in -diversity) and serum SCFAs (8-week change in levels of serum 
SCFAs). 
  
15.4 Data Integrity: All data will be double entered separately by two 
individuals. Blood and stool sample data will be examined for 
computational value errors associated with out-of-range variables. All data 
will be examined for outliers and improbable values. More details provided 
in section 17.1. 
 
16.0 Health Information and Privacy Compliance 
16.1 
Select which of the following is applicable to your research: 
☐ My research does not require access to individual health information.   
☒ I am requesting that all research participants sign a HIPCO approved 
HIPAA 
Disclosure Authorization to participate in the research (either the 
standalone form or the combined consent and HIPAA Authorization). 
☐ I am requesting the IRB to approve a Waiver or an alteration of 
research participant authorization to participate in the research. 
Appropriate Use for Research:  
 
 

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16.2 
Identify the source of Private Health Information you will be using for 
your research (Check all that apply)   
☐ I will use the Informatics Consulting Services (ICS) available through 
CTSI (also referred to as the University's Information Exchange (IE) or 
data shelter) to pull records for me 
☒ I will collect information directly from research participants. 
☐ I will use University services to access and retrieve records from the 
Bone Marrow Transplant (BMPT) database, also known as the HSCT 
(Hematopoietic Stem Cell Transplant) database. 
☐ I will pull records directly from EPIC. 
☐ I will retrieve record directly from axiUm / MiPACS 
☐ I will receive data from the Center for Medicare/Medicaid Services  
☐ I will receive a limited data set from another institution  
☐ Other.  Describe: N/A 
16.3 
Explain how you will ensure that only records of patients who have 
agreed to have their information used for research will be reviewed.  
This information will come only from the participants themselves, with all 
review of any health information occurring in a private room within the 
Epidemiological Clinical Research Center. 
16.4 
Approximate number of records required for review: N/A 
16.5 
Please describe how you will communicate with research participants 
during the course of this research.  Check all applicable boxes 
☐ This research involves record review only. There will be no 
communication with research participants. 
☐ Communication with research participants will take place in the course 
of treatment, through MyChart, or other similar forms of 
communication used with patients receiving treatment.  
☒ Communication with research participants will take place outside of 
treatment settings. If this box is selected, please describe the type of 
communication and how it will be received by participants.  
Participants will be asked their preferred communication method; if 
text and/or email is selected, the appropriate consents will be 
obtained. Diet recalls will be conducted by phone, but only at times 
provided by study participants.  
 
Participants will be contacted only as it pertains to the study 
(scheduling/confirming visits, assessing compliance, troubleshooting 

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technical issues, etc). 
 
Importantly, we will not request access to medical records or any 
other source of private information about the participants. 
 
16.6 
Explain how the research team has legitimate access to 
patients/potential participants: 
 
Potential participants will make the initial contact with study staff. Only 
after this initial contact will study staff reach back out to potential 
participants. 
 
16.7 
Location(s) of storage, sharing and analysis of research data, including 
any links to research data (check all that apply).   
☐ In the data shelter of the Information Exchange (IE)  
 
☐ Store 
☐ Analyze 
☐ Share 
☐ In the Bone Marrow Transplant (BMT) database, also known as the 
HSCT (Hematopoietic Stem Cell Transplant) Database  
 
☐ Store 
☐ Analyze 
☐ Share 
☒ In REDCap (recap.ahc.umn.edu)  
 ☐ Store 
☐ Analyze 
☐ Share 
☐ In Qualtrics (qualtrics.umn.edu) 
 ☐ Store 
☐ Analyze 
☐ Share 
☐ In OnCore (oncore.umn.edu)  
 ☐ Store 
☐ Analyze 
☐ Share 
☒ In the University’s Box Secure Storage (box.umn.edu) 
 ☒ Store 
☐ Analyze 
☒ Share 
☐ In an AHC-IS supported server. Provide folder path, location of server 
and IT Support Contact:  
 ☐ Store 
☐ Analyze 
☐ Share 
☐ In an AHC-IS supported desktop or laptop.  
Provide UMN device numbers of all devices: 
 ☐ Store 
☐ Analyze 
☐ Share 
☒ Other: 

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Indicate if data will be collected, downloaded, accessed, shared or stored 
using a server, desktop, laptop, external drive or mobile device (including a 
tablet computer such as an iPad or a smartform (iPhone or Android 
devices) that you have not already identified in the preceding questions 
☒I will use a server not previously listed to collect/download research data 
We will monitor participants physical activity using the Fitabase® platform. 
Fitabase® is a platform provided by Fitbit that allows for research 
participants to upload their Fitbit smartwatch data each day. We will be 
able to use this platform and an associated walk log to ensure participants 
are engaging in their home-based walking. Importantly, Fitabase® houses 
data in secure, encrypted Microsoft Azure servers and do not sell, release, 
or otherwise make data available to third parties. Further, we will provide 
only de-identified participant IDs to Fitabase®, with Fitabase® only having 
access to de-identified IDs but otherwise unable to access participants’ 
email address or account names. 
☐I will use a desktop or laptop not previously listed 
☐I will use an external hard drive or USB drive (“flash” or “thumb” drives) 
not previously listed  
☐I will use a mobile device such as an tablet or smartphone not previously 
listed 
 
16.8 Consultants. Vendors. Third Parties: N/A 
16.9 Links to identifiable data: Each participant will be assigned a unique study 
ID that will consist of a series of numbers and letters that will have no 
meaning to any of the individual's personal identifying characteristics. The 
investigators will use this ID to track each individual participant in the 
research database. There will be not identifiers in this database. The 
identifiers will be maintained in separate, secure, encrypted, password-
protected database in a different physical location. This separate database 
for the identifying information will also include the study ID so that the 
investigators can link research data to individuals when necessary. 
Electronic data bases, encrypted on University servers and password 
protected, only accessible to the investigators. Limited paper files may be 
maintained in locked filing cabinets in locked offices, but these will be 
minimized and will include consent form copies and a minimal amount of 
participant information if needed in hard copy. No research information 
will be placed in any sort of medical, employment, or educational record. 
 

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16.10 Sharing of Data with Research Team Members: We will share research 
data on a limited basis between research team members. However, when 
we do share data, we will do so via our University Box accounts. 
 
16.11 Storage and Disposal of Paper Documents: Limited paper files may be 
maintained in locked filing cabinets in locked offices near the PI, but these 
materials will be minimized and will include consent form copies and a 
minimal amount of participant information if needed in hard copy. No 
research information will be placed in any sort of medical, employment, or 
educational record. Any paper materials will be kept for a minimum of 3 
years after which the materials will be shredded and disposed of via proper 
means. 
 
17.0 Confidentiality 
17.1 Data Security: As reviewed previously, we have numerous safeguards in 
place to protect data integrity—all of which will be under the oversight of 
the PI. Our research team is qualified to conduct this oversight given past 
experience conducting randomized trials. First, all data will be double 
entered separately by two individuals which will allow us to check for data 
entry errors. Blood, stool, and salivary sample data will be examined for 
computational value errors associated with out-of-range variables. All data 
will be examined for outliers and improbable values. These procedures will 
ensure protection of the study’s internal validity. Second, to protect the 
participants’ health-related information, we will deidentify each 
participant’s data. In detail, and as discussed in section 16.1, each 
participant will be assigned a unique study ID that will consist of a series of 
numbers and letters that will have no meaning to any of the individual's 
personal identifying characteristics. The investigators will use this ID to 
track each individual participant in the research database. There will be not 
identifiers in this database. The identifiers will be maintained in separate, 
secure, encrypted, password-protected database in a different physical 
location. This separate database for the identifying information will also 
include the study ID so that the investigators can link research data to 
individuals when necessary. Electronic data bases, encrypted on University 
servers and password protected, only accessible to the investigators. 
Limited paper files may be maintained in locked filing cabinets in locked 
offices, but these will be minimized and will include consent form copies 
and a minimal amount of participant information if needed in hard copy. 
No research information will be placed in any sort of medical, employment, 
or educational record. 
 
18.0 Provisions to Monitor the Data to Ensure the Safety of Participants 

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This study involves no greater than minimal risk to participants. 
18.1 Data Integrity Monitoring. Our research team is qualified to conduct this 
oversight given past experience conducting randomized trials. First, all data 
will be double entered separately by two individuals which will allow us to 
check for data entry errors. Blood, stool, and salivary sample data will be 
examined for computational value errors associated with out-of-range 
variables. All data will be examined for outliers and improbable values. 
These procedures will ensure protection of the study’s internal validity. 
Second, to protect the participants’ health-related information, we will 
deidentify each participant’s data. In detail, and as discussed in section 
16.1, each participant will be assigned a unique study ID that will consist of 
a series of numbers and letters that will have no meaning to any of the 
individual's personal identifying characteristics. The investigators will use 
this ID to track each individual participant in the research database. There 
will be not identifiers in this database. The identifiers will be maintained in 
separate, secure, encrypted, password-protected database in a different 
physical location. This separate database for the identifying information 
will also include the study ID so that the investigators can link research data 
to individuals when necessary. Electronic data bases, encrypted on 
University servers and password protected, only accessible to the 
investigators. Limited paper files may be maintained in locked filing 
cabinets in locked offices, but these will be minimized and will include 
consent form copies and a minimal amount of participant information if 
needed in hard copy. No research information will be placed in any sort of 
medical, employment, or educational record. 
 
18.2 Data Safety Monitoring. N/A 
 
19.0 Provisions to Protect the Privacy Interests of Participants 
19.1 Protecting Privacy: We will have participants complete the demographic 
questionnaire in a private room well-ventilated within the Epidemiology 
Clinical Research Center. Further, we will conduct all measurements of 
weight, waist circumference, and blood pressure in this same well-
ventilated room within this building as well. These data will not be spoken 
aloud. Finally, all blood collection will occur at the Epidemiology Clinical 
Research Center and performed by an experienced phlebotomist in the 
well-ventilated private room, with the salivary collection occurring within a 
private room in this facility too. 
 

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19.2 Access to Participants: We will only request access to each participant’s 
home address, email, and phone numbers to ensure that we can contact 
participants to set up weekly walking sessions or determine a schedule of 
home-based walking sessions. We will otherwise not request access to 
medical records or any other source of private information about the 
participants. 
 
20.0 Compensation for Research-Related Injury 
20.1 Compensation for Research-Related Injury: This research does not involve 
greater than minimal risk to participants. 
 
20.2 Contract Language: In the event that this research activity results in an 
injury, treatment will be available, including first aid, emergency treatment 
and follow-up care, as needed. Care for such injuries will be billed in the 
ordinary manner to you or your insurance company. If you think that you 
have suffered a research related injury, let the study know right away. 
 
21.0 Consent Process 
21.1 Consent Process (when consent will be obtained): During the recruitment 
process, potential participants will be provided with a copy of the study 
consent form to review. An additional copy will also be attached to their 
consent visit confirmation email/letter for their records. The day prior to 
the first study visit, the study coordinator will call to confirm the visit and 
complete the COVID-19 screening questionnaire. The study coordinator will 
confirm that the participant understands the study goals, design, purpose, 
and risks/benefits and is sure they would like to participate in the study, 
and answer any study questions at that time (to minimize in-person time at 
the ECRC). At the consent visit, the study coordinator will answer any 
additional questions and have the participant sign the official consent form. 
A signed copy of the consent form will be offered to the participant. The 
consent process will take place within a conference room within the 
Epidemiology Clinical Research Center immediately prior to baseline 
testing. We will employ no formal process to ensure ongoing consent, but 
we will continually ensure that the participant is aware of the voluntary 
nature of the study by ensuring the OVPR Participant Information sheet is 
sent 24-hours prior to any in-person visit and knows that they may 
withdraw from study participation at any time for any reason. 
 

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21.2 Waiver or Alteration of Consent Process (when consent will not be 
obtained): N/A 
 
21.3 Waiver of Written/Signed Documentation of Consent (when 
written/signed consent will not be obtained): N/A 
 
21.4 Non-English Speaking Participants: N/A 
 
21.5 Participants Who Are Not Yet Adults (infants, children, teenagers under 
18 years of age): N/A 
 
21.6 Cognitively Impaired Adults, or adults with fluctuating or diminished 
capacity to consent: N/A 
 
21.7 Adults Unable to Consent: N/A 
• 
Permission: N/A 
 
• 
Assent: N/A 
 
• 
Dissent: N/A 
 
22.0 Setting 
22.1 Research Sites: We will identify and recruit participants primarily from the 
health clinics of our clinical partners, but we will also recruit individuals 
from the Minneapolis St-Paul community. We will not be compensating any 
of our clinical partners for their involvement. Research procedures will be 
performed at the Epidemiology Clinical Research Center. 
 
22.2 International Research: N/A 
 
 
23.0 Multi-Site Research 
N/A 
 

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24.0 Coordinating Center Research 
N/A 
  
 
25.0 Resources Available 
25.1 Resources Available:  
• We have clinical partners in Minneapolis-St. Paul health clinics 
which should allow us to complete the recruitment of 100 
participants within two years in addition to our ability to leverage 
University listservs for flier distribution and the posting of fliers 
within local community centers and on social media. We will 
devote 2 years to conducting this research. 
• The West Bank Office Building has ample room for the current 
study. The Division of Epidemiology and Community Health has 48 
faculty, 350 staff, and a combined office space totaling more than 
75,000 square feet. Meeting rooms are equipped with state-of-
the-art video communication technology for communication with 
external consultants and staff working off-site. It is anticipated 
that the current space will be adequate to accommodate the 
needs of this proposal. Office equipment, such as conference 
telephones, printers, photocopiers, and postage services are 
available to the proposed study. Additionally, we already possess 
the two treadmills that will be used to implement the current 
study, with more to be purchased. The Epidemiology Clinical 
Research Center (ECRC) is located one block away from the offices 
of the Division of Epidemiology and Community Health, about 0.5 
miles from the University of Minnesota Hospital on the 
Minneapolis campus, and is readily accessible from all parts of the 
Twin Cities metropolitan area (within three blocks from two major 
interstate highways). Participants in this trial will visit the ECRC 
for the blood draws, saliva sample collection, and other clinic 
measures during the baseline fasting visit and during the 4th and 
8th week time points. Assays from blood draws will be analyzed 
at the University of Minnesota’s Advanced Research and 
Diagnostic Laboratory, with all necessary precautions taken to 
limit any chance of COVID-19 infections (as outlined earlier in 
the protocol). Parking is free. The ECRC occupies one floor 
(17,758 square feet) of a two-story building and includes 
reception area, offices for staff, examination rooms, interview 
rooms, ultrasound, phlebotomy and blood processing rooms, 
freezer room, lounge, conference rooms, and storage space. The 
building meets current regulations for handicapped accessibility 

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and has 100 free adjacent parking spaces. The site was 
established in February 2000 and is currently providing space and 
infrastructure for 20+ research projects including multicenter 
epidemiologic studies and clinical trials 
• We do not anticipate any medical or psychological risk associated 
with participation, but the West Bank Office Building and 
Epidemiology Clinical Research Center has general first aid 
equipment and a defibrillator onsite if any medical needs arise. 
Further, we will ensure that individuals involved in the monitoring 
of study participation are CPR certified. 
• The PI and Co-Is will train any individual assisting with data 
collection procedures/measurements and will supervise these 
individuals performing these procedures/measurements for 1 
week following training. 
 
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Moore S, Lee I-M, Weiderpass E, et al. Association of leisure-time physical 
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3. 
Benjamin E, Muntner P, Alonso A, et al. Heart disease and stroke statistics-2019 
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Koivula R, Tornberg A, Franks P. Exercise and diabetes-related cardiovascular 
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ADA. Economic costs of diabetes in the U.S. in 2017. Diabetes Care. 2018;41:917-
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Afkarian M, Zelnick L, Hall Y, et al. Clinical manifestations of kidney disease 
among U.S. adults with diabetes, 1988-2014. Journal of the American Medical 
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8. 
Young D, Hlvert M-F, Alhassan S, et al. Sedentary behavior and cardiovascular 
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10. 
USDHHS. Physical activity guidelines for Americans: 2nd edition. In. Washington, 
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Schoenborn C, Stommel M. Adherence to the 2008 adult physical activity 
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Kang S, Jeraldo P, Kurti A, et al. Diet and exercise orthogonally alter the gut 
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Liu Z, Liu H-Y, Zhou H, et al. Moderate-intensity exercise affects gut microbiome 
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Petriz B, Castro A, Almeida J, et al. Exercise induction of gut microbiota 
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39. 
Liu T-W, Park Y-M, Holscher H, et al. Physical activity differentially affects the 
cecal microbiota of ovariectomized female rats selectively bred for high and low 
aerobic capacity. PLoS One. 2015;10(8):doi: 10.1371/journal.pone.0136150. 
40. 
Mika A, Van Treuren W, Herrera J, Knight R, Fleshner M. Exercise is more 
effective at altering gut microbial composition and producing stable changes in 
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