Prolonged Daily Fasting as a Viable Alternative to Caloric Restriction in At-Risk Obese Humans
Phase: NA
Conditions: Obesity
Sponsor: University of Minnesota

MEDICAL PROTOCOL (HRP-590) 
PROTOCOL TITLE: Prolonged Daily Fasting as a Viable Alternative to Caloric Restriction in 
At-Risk Obese Humans : SeeFoodStudy2 
 
VERSION DATE: 6/20/2022 
 
Page 1 of 80 
Template Revised On: 09/01/2019 
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  
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Require Scientific Review? Not 
sure? See guidance on next 
page. 
 
Documentation of scientific merit must 
be provided.   
Contact: hrpp@umn.edu  
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☒ No 
Relate to cancer patients, 
cancer treatments, cancer 
screening/prevention, or 
tobacco? 
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(x-ray imaging, 
radiopharmaceuticals, external 
beam or brachytherapy) 
Complete the AURPC Human Use 
Application and follow instructions on 
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committee. 
Contact: barmstro@umn.edu  
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from these 
committees 
must be 
received 
prior to IRB 
approval;  
 
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groups 
each have 
their own 
application 
process.  
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Resonance Research (CMRR) 
as a study location? 
Complete the CMRR pre-IRB ancillary 
review  
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Include the use of 
recombinant or synthetic 
nucleic acids, toxins, or 
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eprotocol.umn.edu 
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Include the use of human fetal 
tissue, human embryos, or 
embryonic stem cells? 
Contact OBAO for submission 
instructions and guidance 

MEDICAL PROTOCOL (HRP-590) 
PROTOCOL TITLE: Prolonged Daily Fasting as a Viable Alternative to Caloric Restriction in 
At-Risk Obese Humans : SeeFoodStudy2 
 
VERSION DATE: 6/20/2022 
 
Page 2 of 80 
Template Revised On: 09/01/2019 
☒ 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  
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☐ 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. 
 
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Laboratory Services to collect 
tissue for research? 
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assigned to your study by IRB staff. 
Contact: cdrifka@umn.edu  
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Have a PI or study team 
member with a conflict of 
interest? 
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assigned to your study by IRB staff 
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☐ No 
Need to be registered on 
clinicaltrials.gov? 
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clinicaltrials.gov ancillary review will be 
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ETHOS, the OnCore ancillary review will 
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Does not 
affect IRB 
approval. 
 
 
 
 

MEDICAL PROTOCOL (HRP-590) 
PROTOCOL TITLE: Prolonged Daily Fasting as a Viable Alternative to Caloric Restriction in 
At-Risk Obese Humans : SeeFoodStudy2 
 
VERSION DATE: 6/20/2022 
 
Page 3 of 80 
Template Revised On: 09/01/2019 
PROTOCOL COVER PAGE 
 
 
 
Protocol Title 
Prolonged Daily Fasting as a Viable Alternative to Caloric 
Restriction in At-Risk Obese Humans 
Principal 
Investigator/Faculty 
Advisor 
Name:  Lisa Chow, MD, MS 
Department: Department: Medicine 
Division of Endocrinology, Diabetes and Metabolism 
Telephone Number: 612-625-8934 
Email Address: chow0007@umn.edu 
Student 
Investigator 
Name: 
Current Academic Status (Student, Fellow, Resident): 
  
Department: 
Telephone Number: 
Institutional Email Address: 
Scientific 
Assessment 
Nationally-based, federal funding organizations 
IND/IDE # (if 
applicable) 
Exempt (IND 147364) 
IND/IDE Holder 
Dr. Lisa Chow 
Investigational Drug 
Services # (if 
applicable) 
Exempt (IND 147364) 
Version 
Number/Date: 
Version 11.0, 6/20/2022 
 

MEDICAL PROTOCOL (HRP-590) 
PROTOCOL TITLE: Prolonged Daily Fasting as a Viable Alternative to Caloric Restriction in 
At-Risk Obese Humans : SeeFoodStudy2 
 
VERSION DATE: 6/20/2022 
 
Page 4 of 80 
Template Revised On: 09/01/2019 
 
REVISION HISTORY 
 
Revision # 
Version Date 
Summary of Changes 
Consent Change? 
1 
05/11/2020 
• 
Removed Overnight Stay, 
• 
Shortened 
hyperinsulinemic-
euglycemic clamp to 4 
hours (previously 6 hours) 
• 
CGM is now optional 
• 
 removed supervised meals 
• 
change U13palmitate to 
D5glycerol (still FDA 
exempt) 
• 
 changed dietary recall to 3 
at baseline and 3 at end 
(rather than 2 at baseline, 2 
at midpoint, 2 at end) 
• 
Reduced compensation to 
$400 due to less participant 
burden 
• 
Added strategies to 
improve recruitment in case 
of recruitment shortfalls 
Yes 
2 
06/30/2020 
• Reduced in person visits 
from 8 to 5 
• 2 visits are virtual (Visit 0 
and Visit 4) 
• Visit 6 and visit 7 are 
combined 
• Insulin resistance inclusion 
criterion has been removed 
• Initial dietician in person 
counseling has been 
changed to a telephone 
counseling session 
• The glucose sensor (CGM) 
will be a study activity 
Yes 
3 
07/27/2020 
• EConsent/HIPAA Form, 
delivered and signed via 
REDCap, will be provided as 
an option for consent 
process dependent upon 
participant preference 
Yes 

MEDICAL PROTOCOL (HRP-590) 
PROTOCOL TITLE: Prolonged Daily Fasting as a Viable Alternative to Caloric Restriction in 
At-Risk Obese Humans : SeeFoodStudy2 
 
VERSION DATE: 6/20/2022 
 
Page 5 of 80 
Template Revised On: 09/01/2019 
• SF-36 survey (Quality of 
Life) added to V3 and V6 
• Added ‘prior to visit fasting’ 
language to a letter we 
send to the participants  
4 
08/17/2020 
• It was clarified in the 
consent form and protocol 
that the fasting labs 
obtained in Visit 1 
determine eligibility.  
• In the protocol, the study 
procedures are made more 
explicit, such as conducting 
the remote/virtual visits 
using Zoom or Doximity. 
• In the consent/HIPAA form, 
consent language has been 
added specific to the use of 
the mCC app. This language 
was provided by the Salk 
Institute to replace Salk’s 
online version of the mCC 
app’s consent that we had 
been previously using.  
• The protocol has been 
updated to reflect the 
addition of the mCC app 
consent language (that Salk 
had been using online and 
they would like us now to 
incorporate into our 
consent form)   to the main 
consent/HIPAA form.  
Yes 
5 
12/22/2020 
• It was clarified in the 
protocol on page 51 the 
study document retention 
requirements the study will 
follow. 
• Additional MRI language 
was added to page 13 of 
the consent form that 
addresses notifing the 
Yes 

MEDICAL PROTOCOL (HRP-590) 
PROTOCOL TITLE: Prolonged Daily Fasting as a Viable Alternative to Caloric Restriction in 
At-Risk Obese Humans : SeeFoodStudy2 
 
VERSION DATE: 6/20/2022 
 
Page 6 of 80 
Template Revised On: 09/01/2019 
participants of significant 
new findings. 
6 
1/5/21 
We have expanded the eligibility 
criteria to include participants with 
at least an 12 hour eating window. 
No 
7 
2/2/21 
We have updated our exclusion 
criterion to reflect the exclusion of 
people on beta blockers or 
medications known to affect 
weight such as TZD, insulin, GLP-1 
agonists, phentermine, or 
sibutamine. Our previous criteria 
of excluding for statin or diuretic 
use has now been removed as this 
will not affect our outcome 
parameters as initially believed.  
Changing this criteria will not 
increase risk and will broaden our 
participant pool. 
No 
8 
2/18/2021 
We are including ResearchMatch 
as a recruitment tool. We have 
also updated the calculations for 
the continuous infusion of 
D5glycerol (a naturally occurring 
and stable isotope) and the timing 
of the bolus for D5 glycerol and 6,6 
2H2 glucose performed during V3 
and V6. This does not impact 
participant safety. This is to 
accommodate for slight variability 
in the compounding by the 
compounding pharmacy and 
available pumps at CRU.  
No 
9 
8/31/2021 
To increase retention in the non-
TRE (control) group, we will offer a 
complimentary dietary session 
after the intervention and Visit 6 
have been completed to point out 
dietary changes the participant 
may wish to consider to support 
good nutrition and health body 
No 

MEDICAL PROTOCOL (HRP-590) 
PROTOCOL TITLE: Prolonged Daily Fasting as a Viable Alternative to Caloric Restriction in 
At-Risk Obese Humans : SeeFoodStudy2 
 
VERSION DATE: 6/20/2022 
 
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weight. A resource book may also 
be provided. 
10 
6/20/2022 
We are eliminating the inclusion 
criterion “Self-reported absence of 
known sleep apnea” since it is not 
scientifically relevant to our study 
aims and by eliminating it, we will 
be able to expand eligibility. 
No 
 
 
 

MEDICAL PROTOCOL (HRP-590) 
PROTOCOL TITLE: Prolonged Daily Fasting as a Viable Alternative to Caloric Restriction in 
At-Risk Obese Humans : SeeFoodStudy2 
 
VERSION DATE: 6/20/2022 
 
Page 8 of 80 
Template Revised On: 09/01/2019 
Table of Contents 
 
1.0 
Objectives 
9 
2.0 
Background 
11 
3.0 
Study Endpoints/Events/Outcomes 
17 
4.0 
Study Intervention(s)/Investigational Agent(s) 
17 
5.0 
Procedures Involved 
18 
6.0 
Data and Specimen Banking 
37 
7.0 
Sharing of Results with Participants 
39 
8.0 
Study Population 
39 
10.0 Local Number of Participants 
42 
11.0 Local Recruitment Methods 
42 
12.0 Withdrawal of Participants 
44 
13.0 Risks to Participants 
45 
14.0 Potential Benefits to Participants 
48 
15.0 Statistical Considerations 
48 
16.0 Health Information and Privacy Compliance 
50 
17.0 Confidentiality 
54 
18.0 Provisions to Monitor the Data to Ensure the Safety of Participants 
54 
19.0 Provisions to Protect the Privacy Interests of Participants 
57 
20.0 Compensation for Research-Related Injury 
57 
21.0 Consent Process 
57 
22.0 Setting 
59 
23.0 Multi-Site Research 
64 
24.0 Coordinating Center Research 
64 
25.0 Resources Available 
64 
26.0 References 
64 
 

MEDICAL PROTOCOL (HRP-590) 
PROTOCOL TITLE: Prolonged Daily Fasting as a Viable Alternative to Caloric Restriction in 
At-Risk Obese Humans : SeeFoodStudy2 
 
VERSION DATE: 6/20/2022 
 
Page 9 of 80 
Template Revised On: 09/01/2019 
ABBREVIATIONS/DEFINITIONS 
• 
BMI (body mass index)  
• 
Co-I (co-investigator)   
• 
CR (caloric restriction)  
• 
CGM (continuous glucose monitoring)  
• 
CMRR (Center for Magnetic Resonance Research)  
• 
DXA (Dual X-ray absorptiometry)  
• 
D5glycerol (D5glycerol with 5 hydrogens labeled with deuterium – a stable 
 
isotope 
• 
EHR (electronic health record)   
• 
FFA (free fatty acid)  
• 
FFM (fat free mass)  
• 
H: Hypothesis  
• 
HFF (hepatic fat fraction)  
• 
HbA1c (hemoglobin A1c)  
• 
HOMA-IR (homeostatic model of insulin-resistance)  
• 
mCC (MyCircadianClock)  
• 
MRI (magnetic resonance imaging)  
• 
MRS (magnetic resonance spectroscopy)  
• 
NAFLD (non alcoholic fatty liver disease)  
• 
NCC (Nutrition Coordinating Center),  
• 
NDSR (Nutrition Data System for Research_  
• 
Non-TRE (non time restricted eating)  
• 
OGTT (oral glucose tolerance test)  
• 
mCC (my Circadian Clock)  
• 
QOL (quality of life)  
• 
RQ (respiratory quotient)  
• 
SD (Standard deviation)  
• 
TRE (time restricted eating)   
• 
U13C palmitate (palmitate with all carbons labeled with 13C -  a stable isotope)  
• 
UMMC - University of Minnesota Medical Center 
• 
WT (wild type)  
• 
VO2max (maximum oxygen consumption)  
  
 
 

MEDICAL PROTOCOL (HRP-590) 
PROTOCOL TITLE: Prolonged Daily Fasting as a Viable Alternative to Caloric Restriction in 
At-Risk Obese Humans : SeeFoodStudy2 
 
VERSION DATE: 6/20/2022 
 
Page 10 of 80 
Template Revised On: 09/01/2019 
 
1.0 
Objectives 
1.1 Purpose: Obesity is reaching epidemic proportions, affecting 36% of the adult 
population in the United States. There is intense interest in dietary management to treat 
obesity and its associated complications. The first line of obesity treatment is caloric restriction 
(CR), although recidivism is common. For moderate CR, attrition rates of 20% are often 
reported, therefore weight loss options beyond CR are urgently needed.  
Time restricted eating (TRE) shifts the paradigm in obesity treatment. TRE arose from 
synergizing food intake with the circadian rhythm. In animals, this meant limiting food intake 
(~ 8 hours, isocaloric) to their active phase, resulting in many metabolic benefits (proportional 
to fasting duration) such as reduction in adiposity, liver steatosis, hyperlipidemia and insulin 
resistance compared to mice fed ad libitum.  
In America, humans have constant access to food. As the majority of Americans are 
perpetually eating (median eating window:14.8 hours), TRE presents another approach 
towards obesity treatment. In humans, TRE imposes a daily eating window (~8-10 hours) 
resulting in a daily 14-16 hour fast. Current human TRE studies report loss of weight and fat 
mass, with equivocal effects on glucose metabolism. These studies have limitations, including 
lack of randomization, restriction to single-sex, or provision of all food (limiting translation).  
We performed a pilot study randomizing healthy, overweight (≥25 kg/m2) participants with 
an eating window of ≥14 hours to either TRE with ad libitum intake (n=11, self-selected 8 hour 
window) or unrestricted eating (non-TRE: n=9) for 12 weeks (Preliminary data). All oral intake 
was documented (image, time) using a mobile phone app with good compliance [Mean (SD): 
86.6% (11.8) of days]. The TRE group narrowed their eating window to 9.9 hours (2.0), a 
significant decline of 5.4 hours (2.2) from baseline (p<0.0001). In contrast, the non-TRE group 
did not significantly alter their eating window. At end-intervention, TRE reduced weight [-3.7% 
(1.8)], fat mass [-4.0% (2.9)], visceral fat [-11.1% (13.4)], fasting glucose [-7.7% (6.9)] and 
fasting triglyceride levels [-23.6% (21.7)] relative to baseline (all p<0.05). These measures were 
unchanged in non-TRE. Several key findings were seen: 1) TRE reduced weight, fat mass and 
visceral fat compared with non-TRE; 2) Greater restriction of the eating window was 
associated with greater fat mass/visceral 
fat loss; 3) TRE improved self-reported 
quality of life, 4) TRE did not alter 
actigraphy measured physical activity or 
sleep measures relative to baseline or the 
non-TRE. Therefore, although TRE-
associated benefits are conventionally 
believed to be due to matching eating with circadian rhythms, our data challenge this notion 
and suggest that prolonged daily fasting as the key determining factor.  
We hypothesize that TRE with ad libitum intake presents a highly viable alternative to CR 
in reducing weight, fat mass and improving insulin sensitivity, as daily prolonged fasting 
reduces caloric intake and enhances metabolic flexibility—the adaptability of fuel selection 
given ambient fuel exposure. (Fig 1: Conceptual model). We will recruit obese, insulin-

MEDICAL PROTOCOL (HRP-590) 
PROTOCOL TITLE: Prolonged Daily Fasting as a Viable Alternative to Caloric Restriction in 
At-Risk Obese Humans : SeeFoodStudy2 
 
VERSION DATE: 6/20/2022 
 
Page 11 of 80 
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resistant humans (n=72, 24/group) with an eating window ≥14 hours. Participants will be 
randomized to 1 of 3 groups: 1) TRE (8 hour eating window) with ad libitum intake, 2) CR with 
15% reduction of caloric intake, 3) non-TRE with ad libitum intake for 12 weeks. Measures 
performed pre-post intervention will address the following Aims: 
Aim#1: Evaluate the effect of TRE with ad libitum intake on weight and body 
composition. H 1.1: Individuals in the TRE and CR groups will have similar weight loss, which 
will be greater than weight loss achieved in the non-TRE group (primary outcome). H 1.2: TRE 
will result in greater loss of loss of total body fat (quantified by DXA) and greater loss of 
hepatic/visceral fat/ectopic fat (quantified by MRI) than CR. 
Aim#2: Assess the effect of TRE with ad libitum intake on caloric balance. H 2.1: TRE will 
reduce caloric intake compared with non-TRE [gold-standard interviewer administered 24-
hour dietary recall (primary outcome)] with similar reduction as with CR, H.2.2: TRE will not 
alter physical activity, but will increase fat oxidation compared with CR and non-TRE. 
Aim#3: Assess the effect of TRE with ad libitum intake on metabolic flexibility. H 3.1: TRE 
will enhance metabolic flexibility compared with CR and non-TRE as measured by indirect 
calorimetry [RQ:Respiratory quotient before and during 2 step 6,6-2H2 hyperinsulinemic-
euglycemic clamp: primary outcome].  H 3.2: TRE will improve insulin sensitivity compared 
with non-TRE and similar to CR. H 3.3: TRE will augment greater fasting lipolysis compared to 
CR and non-TRE as measured by D5glycerol and enhance lipolysis suppression during the 2 
step 6,6-2H2 hyperinsulinemic-euglycemic clamp. 
If our hypotheses are confirmed, this project has significant impact. First, it will advance 
our understanding of the mechanisms underpinning this innovative intervention. Second, TRE 
can be a practical means of implementing prolonged fasting on a large scale, thereby 
transforming the treatment of obesity. 
 
2.0 
Background 
2.1 Significance of Research Question/Purpose:  
Caloric restriction is the mainstay of obesity treatment, alternative options need to be 
considered Obesity is reaching epidemic proportions, affecting 36% of the adults in the United 
States,2 so there is intense interest in dietary management to treat obesity and its 
complications. The first line of treatment is caloric restriction (CR), although recidivism is 
common.3 For moderate CR, attrition rates of 20% are reported, with most participants 
returning to their baseline weight within 5 years of completing therapy. Therefore, sustainable 
weight loss options beyond CR are urgently needed. 
Constant eating is unhealthy in animal models, yet humans constantly eat. Mice 
permitted ad libitum intake and continuous access to high fat/high sugar food experience 
adverse metabolic effects, such as obesity, hyperinsulinemia, hepatic steatosis, and 
inflammation.1,2 Yet, when these mice were allowed ad libitum intake only during a restricted 
eating window (TRE: ~8-9 hours window during the dark phase), marked reductions in 
adiposity, liver steatosis, hyperlipidemia, insulin resistance and glucose tolerance were 
observed despite similar energy intake compared with their unrestricted counterparts. The 

MEDICAL PROTOCOL (HRP-590) 
PROTOCOL TITLE: Prolonged Daily Fasting as a Viable Alternative to Caloric Restriction in 
At-Risk Obese Humans : SeeFoodStudy2 
 
VERSION DATE: 6/20/2022 
 
Page 12 of 80 
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metabolic benefits associated with TRE were proportional to the fasting duration1 and suggest 
that adverse effects of diet can be disassociated from diet composition and caloric intake.  
Similarly, humans in modern America have constant access to food. Not surprisingly, 
humans are perpetually eating. Dr. Panda (Co-I) who developed myCircadianClock (mCC), a 
smartphone app, to track food intake.3 In a recent study (n=156 for 3 weeks, all food intake 
documented by the mCC app), he reported that humans have frequent and erratic eating 
patterns, with more than half of adults eating ≥15 hours per day and only stopping to eat 
when asleep ( <1% of eating events between 1-6 am).3  
As constant eating, rather than diet composition, drove the metabolic complications in 
mice,1,2 constant eating, in the form of snacking, likely contributes to adverse metabolic 
complications in humans. In adults, snacking has increased in terms of number of snackers 
(1977-1978:60% of the population, 2009-2012: 86% of the population) and higher caloric 
intake from snacks (1977-1978:~200 kcal/day, 2009-2012: ~ 500 kcal/day).4   
 Time restricted eating studies in humans suggest metabolic benefits Time restricted 
eating (TRE) presents an alternative approach to CR. TRE imposes a consistent daily eating 
window (~8-10 hours) with ad libitum intake while preserving a daily 14-16 hour fasting 
period. By focusing on restricting time rather than restricting calories, TRE is a practical 
means of implementing prolonged fasting on a large scale in the population.  
Not surprising, human TRE studies are promising3,5,6 but preliminary, with limitations 
including lack of randomized control3,6 or restriction to men in an environment where all food 
was provided.5 
Generally, the TRE studies with ad libitum intake have shown TRE facilitates weight loss 
relative to baseline.3,6 TRE studies with isocaloric intake maintain weight, with consistent fat 
mass loss,7,8 and variable effects on fasting glucose,[decrease7 no change8] fasting 
insulin[decrease5,7], fasting triglycerides [decrease7 no change8 increase5], insulin sensitivity 
[increase5] and β cell responsiveness [increase5]. 
To date, there are no published randomized, trials using TRE as an intervention to both 
sexes in the community setting. Hence, we performed a pilot study (Preliminary data, 
submitted for publication), a randomized clinical trial examining the effect of TRE vs non-TRE 
for 12 weeks on body composition, lipids and glycemic measures in overweight/obese humans 
without diabetes. Our key findings include: 1) Compared to non-TRE, TRE decreased weight, 
total fat mass, lean mass and visceral fat (all p≤0.05),  Compared to pre-intervention, TRE 
reduced weight, fat mass, lean mass, visceral fat, fasting glucose, and fasting triglyceride 
levels. 2) Greater restriction of the eating window was associated with greater fat 
mass/visceral fat loss. 
TRE appears to be sustainable. One study3 (n=8) enrolled humans with ≥14 hour eating 
window for a 16 week TRE intervention (10-12 hours eating window). Post-intervention, the 
participants reduced their eating window by ~4.5 hours and lost weight (~ 3.3 kg). Afterwards, 
all participants continued with TRE for 36 more weeks. At study conclusion (Week 52), these 
participants maintained their weight loss (~ 3.3 kg) and reported improved energy, and sleep 
compared with baseline. Our preliminary data also reports TRE improving quality of life 
relative to baseline and the non-TRE group (Section 3.3.5). To date, there are no published 

MEDICAL PROTOCOL (HRP-590) 
PROTOCOL TITLE: Prolonged Daily Fasting as a Viable Alternative to Caloric Restriction in 
At-Risk Obese Humans : SeeFoodStudy2 
 
VERSION DATE: 6/20/2022 
 
Page 13 of 80 
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studies comparing sustainability of TRE to CR. Yet, CR is associated with attrition rates of 20-
30%.9-11 With pilot studies demonstrating TRE improving self-reported quality of life, TRE may 
be a viable option to CR. 
Mechanisms driving TRE need to move beyond matching to circadian rhythm  The concept 
of TRE arose within the context of circadian rhythms. Circadian rhythms are daily ~ 24 hour 
rhythms observed in organisms. These rhythms are at the central level (suprachiasmatic 
nucleus-regulated by the light/dark cycle) as well as at the peripheral level (tissue specific- 
regulated by food intake).12 In mice, restricting the feeding window to the dark cycle, where 
mice are most active, provides metabolic benefits compared with mice fed ad libitum despite 
similar diet composition and energy caloric intake.1,2  
In humans, there is much interest in the clinical significance of TRE timing. Specifically, 
eating earlier in the day is believed to synchronize the central/peripheral clocks to improve 
metabolic outcomes.12 Cross-sectional studies have shown that eating earlier in the day is 
associated with lower body fat than eating later in the day.13,14Clinical trials have 
demonstrated that higher caloric intake at breakfast (n=96, diet=1400 kcal/day * 12 weeks 
with 50% of calories consumed at breakfast or dinner) 15 or lunch (n=80, diet=2000 kcal/day * 
12 weeks with 50% of calories consumed at lunch or dinner)16 results in greater weight loss 
(1.3-2.5 fold) than higher calorie intake at dinner despite similar daily caloric intake.  
Early eating may also improve glucose metabolism, as glucose metabolism appears to be 
under circadian regulation.17  Oral glucose tolerance testing (OGTT) performed at either 7 AM 
or 7 PM demonstrated worse glucose tolerance (70% higher glucose) at 7 PM.18  In men with 
prediabetes (n=13, 6 hour TRE with all food consumed before 2 pm vs 12 hour intake), early 
TRE with isocaloric intake did not alter OGTT glucose levels but reduced OGTT insulin levels by 
50% compared with isocaloric intake over 12 hours.5 Insulin sensitivity (improved by 15%) and 
glucose tolerance (improved by 7%), are improved when the majority of calories are consumed 
during breakfast than supper.19 Yet, in obese, insulin resistant men, the timing of the meal  
[n=23, 4 week with hypocaloric diet (50% of caloric need) with 50% of daily energy consumed 
either at breakfast or dinner] was unrelated to weight loss, hepatic and peripheral insulin 
sensitivity (hyperinsulinemic-euglycemic clamp), intrahepatic triglyceride content, or resting 
energy expenditure.20 Therefore, although early meal timing may be more advantageous over 
later meal timing, the differences are not dramatic.  
Moreover, Dr. Panda (Co-I) recently showed that TRE protected circadian mutant mice from 
the consequences of a high fat diet (weight gain, fat accumulation, lipid profile, glucose 
intolerance, insulin resistance) similar to WT mice,21 supporting the concept that TRE can 
disassociated from the circadian clock in providing metabolic benefits.  As the evening meal 
has a significant social component, its inclusion in any dietary intervention will promote 
sustainability. In our preliminary data, participants self-selected their 8 hour eating window to 
intentionally include the evening meal. Therefore, timing of meals relative to the circadian 
rhythm is desirable, it is not imperative and may be difficult to sustain in practice. 
 Metabolic benefits are seen with fasting In animals and humans, fasting interventions are 
associated with metabolic benefits, including reduced weight, fat mass and insulin 
resistance.12 In humans, fasting interventions with benefits include intermittent fasting (low 

MEDICAL PROTOCOL (HRP-590) 
PROTOCOL TITLE: Prolonged Daily Fasting as a Viable Alternative to Caloric Restriction in 
At-Risk Obese Humans : SeeFoodStudy2 
 
VERSION DATE: 6/20/2022 
 
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calorie intake 1-2 days/week, ad libitum intake 5 days/week),22 fasting mimicking diets (5 day 
regimen which mimic water-only fasting with a micronutrient content which maximizes 
nourishment- performed once per month)23 and most recently TRE.3  Prolonged fasting 
mobilizes triglycerides from the adipose tissue, with 60% of the increase in lipid kinetics 
occurring between 12-24 hours of fasting.24 In contrast, CR can be disassociated from fasting. 
CR in the form of constant eating, even in small amounts, raises serum insulin levels to inhibit 
lipolysis, promotes glucose utilization, and reduce lipid oxidation.25   
This project’s objective is to examine the role of prolonged fasting in TRE-associated 
metabolic changes. The scientific premise is that prolonged fasting associated with TRE 
reduces caloric intake and enhances metabolic flexibility—the adaptability of fuel selection 
given ambient fuel exposure. Therefore, TRE will enhance fat oxidation in the fasting state and 
carbohydrate oxidation in the fed state, to be more metabolically advantageous than CR 
without prolonged fasting. The significance is establishing TRE as a viable alternative to CR, 
potentially transform the treatment of obesity and its associated complications 
2.2 Preliminary Data:  
We performed a pilot study (October 2017-December 2018) randomizing overweight 
participants without self-reported diabetes and a prolonged eating window (≥14 hours) to 
either TRE with ad libitum intake (n=11: 8 hour eating window) or unrestricted eating (n=9: 
non-TRE) for 12 weeks. A baseline period [mean (SD): 28.5 days (8.8)] was need to establish 
the eating window [15.4 hours (0.9)] and to obtain CGM and actigraph data (up to 2 weeks) All 
oral intake was logged using a mobile phone app (MCC).3  Adherence/logging issues were 
assessed weekly and participants contacted if issues were identified. TRE did not alter 
actigraphy-measured physical activity (% time in sedentary, light, moderate or vigorous 
activity) or sleep measures (Pittsburgh sleep quality index, Epworth sleepiness scale, sleep 
duration, sleep efficiency, wake or sleep time) compared to baseline or non-TRE. Key findings 
include the following: 
Preliminary data: TRE alters body composition relative to non-TRE  
Rationale: We examined the effects of TRE on 
weight compared with non-TRE, as well as the 
effects of TRE on body composition 
(determined by DXA) compared with non-TRE.  
Results:  Compared to the non-TRE group, TRE 
lost weight, visceral fat, and lean mass (all p< 
0.05: Fig 2). The TRE group significantly 
reduced weight [Mean (SD): -3.7% (1.8)], fat 
mass [-4% (2.9)], lean mass [-3.0% (2.7)], 
visceral fat [11.1% (13.4)], fasting glucose [-
7.7% (6.9)] and fasting triglyceride levels [-

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23.6% (21.7)] compared to baseline. Given the observed weight loss of 1.5 kg (1.3) lean mass, 
1.6 kg (1.5) fat mass and 0.26 kg (0.4) visceral fat over 12 weeks, consumption of ~270 calories 
less per day may explain the weight loss. Lean mass loss was inversely associated with higher 
baseline body fat mass and insulin resistance. 
Relevance: TRE favorably alters body composition relative to non-TRE. The next steps include 
1) Comparison of TRE vs CR effects on body composition (Aim 1), 2) Using MRI to evaluate TRE 
effects on region-specific fat changes (hepatic, visceral, ectopic) (Aim 1), 3) Detailed evaluation 
of TRE with ad libitum intake on caloric intake using multiple objective measures (Aim 2).  
 
 Preliminary data:  Greater restriction of the TRE eating window is associated with 
greater improvement in body composition  
Rationale: The metabolic effects of TRE likely depends on compliance. Therefore, we were 
interested in the relationship between eating window restriction and weight/body 
composition pre-post intervention.  
Results:   Prior to the intervention, we found that 
BMI was positively associated with eating window 
duration (Fig 3). In the setting of TRE, greater 
restriction of the eating window was moderately 
associated with greater loss of fat mass (Fig 4, Panel 
A) and significantly associated with greater loss of 
visceral fat (Fig 4, Panel B).   
Relevance:  Given no change in actigraphy-measured 
sleep measures, these results suggest that restricting 
the eating window, which prolongs the daily fasting 
period, may drive TRE associated changes in body 
composition.  
 
Preliminary data: TRE 
improves metabolic outcomes  
Rationale:  Evaluate the effects 
of TRE on metabolic measures in 
overweight/obese humans 
without self-reported diabetes 
and an eating window≥ 14 
hours. 
Results: Compared to baseline, 
the TRE group reduced fasting 
glucose [-7.7% (6.9)] and fasting 
triglyceride levels [(-23.6% 
(21.7)] (all p≤0.05). HOMA-IR, Matsuda index, HbA1c did not change. The non-TRE group did 
not change. 

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Relevance: Metabolic benefits are observed with TRE in obese humans without self-reported 
diabetes. TRE may benefit participants with insulin resistance or prediabetes.  
 
 Preliminary data: Metabolic effects of TRE are observed despite “real-world” compliance 
Rationale:  Using prospective documentation of food intake during the baseline and 
intervention by the mCC app, we characterized logging and TRE/non-TRE adherence.   
Results:  The baseline eating window was 15.4 hours (0.9). All participants logged adequately, 
as demonstrated by logging two meals ≥ 5 hours apart on a given day, during the 12 week 
study [86.6% (11.8) of days]. The TRE group narrowed their eating window to 9.9 hours (2.0), a 
significant (p<0.001) decline of 5.4 hours (2.2) from baseline and compared with non-TRE 
[decline of 0.4 hours (1.0)]. The average TRE eating window was from 10:40 AM(54 min) to 
6:40 PM (54 min). The TRE group were ±15 minutes of their designated eating window for 
55.5% (22.4) of days and  ±1 hour of their eating window for 66.3% (20.7) of days. 
Relevance: Participants were able to successfully log their food intake using the mCC app for 
12 weeks. Despite the instruction of limiting TRE to 8 hours, the TRE participants realistically 
reduced their eating window to 10 hours. TRE is still efficacious despite “real-world” 
compliance.  
 
Preliminary data: TRE is an acceptable intervention  
Rationale: Evaluate the impact of TRE on participant self-reported quality of life (QOL) using 
the short form health form questionnaire (SF-36)   
Results: In the TRE group limitations due to Emotional Health improved from baseline (p=0.02) 
and relative to the non-TRE Group (p=0.02). In the TRE group, perception of change in health 
within the last year also improved relative to baseline (p=0.01) and the non-TRE group 
(p=0.005).  
Relevance: TRE improves QOL and may be a viable alternative option to CR.  
 
Metabolic flexibility from training enhances fasting lipolysis  
Rationale: Use stable isotopes of palmitate (U13C 
palmitate, D9palmitate) to measure lipolysis (Dr. 
Chow’s R01) with prolonged fasting (14 hours 
fasting, 20 hours fasting) in lean, trained [VO2 
max:56 ml/kg/min (3.2] subjects and obese, 
sedentary [VO2max: 27 ml/kg/min (4)], insulin 
resistant subjects (Fig 5).  
Results: While fasting, lipolysis is higher in trained 
participants than obese, insulin resistant 
participants. 
Relevance: This data shows several key points: 1) 
feasibility in measuring lipolysis with stable 
isotopes, 2) During prolonged fasting, trained 

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participants have higher lipolysis rates, reflecting higher fat utilization, than overweight, 
sedentary, insulin resistant participants. 
 
2.3 Existing Literature: This is extensively discussed in Section 2.1 
3.0 
Study Endpoints/Events/Outcomes 
3.1 Primary Endpoint/Event/Outcome:  
● The primary outcome for the study is the primary outcome in Aim 1: 
weight loss from baseline to 12 weeks. This was used to determine 
sample size. 
3.2 Secondary Endpoint(s)/Event(s)/Outcome(s):  
● The secondary outcome for Aim 1 is change in body composition from 
baseline to week 12. 
● The primary outcome for Aim 2 is change in caloric intake from baseline 
to week 12.  
The primary outcome for Aim 3 is metabolic flexibility from baseline to week 12. 
4.0 
Study Intervention(s)/Investigational Agent(s) 
4.1 Description:  
Intervention: 
To equalize the intervention exposure, the TRE, CR, and non-TRE groups will 
receive an initial telephone counseling session with the study dietitian followed 
by telephone counseling sessions (including mCC logging) weekly*4 weeks, then 
every 2 weeks. The TRE and CR groups will receive counseling on eating 
window+mCC loggin (TRE) or dietary changes+mCC logging (CR). The non-TRE 
group will receive only the initial counseling which will address themCC logging. 
Separately, weekly feedback/counseling on the participants’ mCC logging will be 
given via email or text message (participant preference) to all participants in the 
TRE, CR, and non-TRE groups.  
Time Restricted Eating (TRE)   
For the TRE group, we will restrict the eating window to 8 hours, where they 
will eat ad libitum. This is the same interval established by Dr. Panda3 and by our 
preliminary data. This interval will be entered into the mCC app and participants 
will be asked to adhere to this eating window during the intervention. All eating 
occasions will be logged using the mCC app. Only water and medications will be 
allowed outside of the eating window.  
 
Caloric Restriction (CR) 

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Participants randomized to CR will meet with the study dietitian prior to the 
intervention and be counseled on options to reduce their caloric intake by 15%, 
while maintaining their eating window. The 15% reduction was selected as our 
preliminary data and recent literature6 suggest that TRE with ad libitum intake 
reduces caloric intake by ~270 to 300 cal/day. The  15% CR  is similar to the 11.9% 
CR achieved by the CALERIE-2 study, which is a 2 year study of CR.26  All eating 
occasions will be logged using the mCC app. The weekly dietitian review of the 
mCC information will include maintenance of the eating window and examination 
of dietary intake to determine compliance with the 15% CR.  
 
Unrestricted Eating (non-TRE) 
For the unrestricted eating (non-TRE) group, participants will eat ad libitum 
per their usual habits. They will receive initial counseling about mCC logging. All 
eating occasions will be logged using the mCC app.  
 
4.2 
IND exemption 
An IND exemption is requested from the FDA as we will be using stable isotopes 
of glucose and fatty acids to trace glucose and lipid metabolism respectively in 
the body. 
4.3 Drug/Device Handling:  
Labeled D5Glycerol and 6,6,2H2  glucose (sterility and pyrogen-free certified) will 
be used for infusions during the clamp procedures and prepared by the Fairview 
Investigational Drug Services (IDS) pharmacy. They will be purchased from 
Cambridge Isotopes (Tewksbury, MA), an established vendor for stable isotopes. 
The D5Glycerol measures lipid kinetics using tracer doses. The 6,6,2H2 glucose 
measures metabolism using tracer doses and glucose disposal. We will employ 
MS/MS approaches to verify the purity of the isotope after purchase. 
4.4 
Biosafety: N/A  
4.5 
Stem Cells: N/A 
4.6 
Fetal Tissue: N/A 
 
5.0 
Procedures Involved 
5.1 Study Design: 
Fig 6 shows the study design. Our strategy is to test the metabolic effects of 12 
week TRE (8 hour feeding window), CR (at 15% intake) and unrestricted eating 
(non-TRE). We will enroll adults with BMI ≥30 kg/m2 who eat over ≥12 hours per 
day.  Pre and post intervention, we will examine the effect of feeding window 
restriction on weight, caloric balance (caloric intake, activity), body composition 

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[DXA,MRI] and metabolic flexibility [indirect calorimetry, D5Glycerol to measure 
lipolysis, 6,6 2H2 hyperinsulinemic-euglycemic clamp].  
 
 

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After the baseline assessment, the participants will be randomized to one of 3 
dietary interventions; 1) TRE (8 hour feeding window), 2) CR (at 15% intake) and 
unrestricted eating (non-TRE). All oral intake will be logged using the mCC phone 
application and reviewed weekly with the participants. We hypothesize that TRE 
with ad libitum intake presents a highly viable alternative to CR in reducing 
weight, fat mass and improving insulin sensitivity, as daily prolonged fasting 
reduces caloric intake and enhances metabolic flexibility—the adaptability of 
fuel selection given ambient fuel exposure.  
The study design is shown in Fig 6. Visits 1-3 will be completed within 3-5 weeks. 
Visits 5 and 6 will be completed within 2-3 weeks post-intervention. Assessment 
details are noted under each Aim. 
5.2 Study Procedures:  
Timeline  
Each participant will be in the study for about 16 weeks with 6 phone visits, 2 
remote/virtual visits (visits 0 and 4) and 5 in-person visits at the University of 
Minnesota (visits 1, 2, 3, 5, 6). Zoom (HIPAA-secure communciations platform used 
by the University of Minnesota) or Doximity (a HIPAA-secure communication 
platform used by healthcare professionals) will be used to conduct the 
remote/virtual visits. Econsent and surveys will be delivered via REDCap to the 
participant during the remote/virtual visits. Two of the 5 in-person visits (visit 3, 
visit 6) will be prolonged (~ 8 hours)  due to the hyperinsulinemic-euglycemic 
clamp.  
 
Intervention:  
Randomized by computer generated code to 1 of 3 groups for a 12 week intervention. A 
dietician will provide the initial dietary telephone counseling to the participants in all 3 groups 
after Visit 3. Only, the TRE and CR groups will receive dietary counseling weekly for 1st 4 weeks 

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and every 2 weeks thereafter. Participants in all 3 groups will receive mCC logging feedback 
involving weekly email/text (participant preference) on mCC logging starting after Visit 1. 
  
• 
Time Restricted Eating (TRE)    
For the TRE group, we will restrict the eating window to 8 hours, where they are 
allowed unrestricted eating within this window. This interval will be entered into 
the mCC app and participants will be asked to adhere to this eating window during the 
intervention. All eating occasions will be logged using the mCC app. Only water and 
medications will be allowed outside of the eating window.   
  
• 
Caloric Restriction (CR)  
Participants randomized to CR will meet with the study dietitian prior to the 
intervention and be counseled on options to reduce their caloric intake by 15%, while 
maintaining their eating window.  All eating occasions will be logged using 
the mCC app. The weekly dietitian review of the mCC information will include 
maintenance of the eating window and examination of dietary intake to determine 
compliance with the 15% CR.   
  
• 
Unrestricted Eating (non-TRE)  
For the non-TRE group, participants will eat per their usual habits. They will receive 
initial counseling about mCC logging with routine feedback . All eating occasions will be 
logged with mCC app.   
  
Procedures  
Height/Weight at Clinical Research Unit (CRU) /Delaware Clinical Research Unit 
(DCRU) 
Height and weight (primary outcome) will be measured at Visit 0 (self-report),  Visit 
1-3, Visit 4 (self-report), Visit 5-6.  
  
Surveys 
A 5 question screen will be conducted at the telephone visit to screen for eating 
disorders (see attached sheet).27 Additional Surveys will be conducted at Visits 1,4,5 
to assess participant’s perception of appetite and at Visits 3 and 6 to assess a 
participant’s quality of life.28  A follow up survey (optional for participant) will be 
sent by email to Redcap line (1 month, 3 months, and 6 months) after the 
intervention to assess ongoing participation in either TRE or CR. 
 
myCircadianClock (mCC) application  
My Circadian Clock (Fig 7) : This smartphone app was designed and is 
currently maintained by Dr. Panda‘s group. The app is freely available to download 
for the iPhone or Android platform and allows a participant to use the smartphone 
camera to take a picture of the specific food or beverage prior to eating. The time 
stamp and the location are noted transferred de-identified to a data server, which 

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is linked back to the individual by the study team. Participants are also reminded at 
random times (1-2 times per day) to input recent food intake.  The mCC data will be 
used to determine study eligibility between Visit 0 and Visit 1. Participants who are 
eligible for the study will continue to use the mCC app for the remainder of the 
study. 
  
Sample collection at Clinical Research Unit (CRU)  
Blood drawn at Visit 1 will be outside the clamp study visits and 
will be drawn in a fasting state (at least 8 hours from last-reported 
non-water intake). Blood at Visits 3 and 6 will be drawn in 
conjunction with the clamp procedure. 
  
  
Visit 1:  Blood will be drawn to measure glucose and insulin 
and for complete metabolic panel, hemoglobin (Hgb), 
thyroid function, lipid panel and  HbA1c as well as whole 
blood (5 ml) saved serum (5 ml)  and saved plasma (5 ml) 
to determine eligibility. Anticipated total amount of blood 
drawn at 45 ml or 3 tablespoons. Participants will be provided the results of 
these tests.  
 
Visit 3: Blood will be drawn to determine fatty acid lipolysis and insulin 
sensitivity. Baseline blood samples will be taken to measure glucose, FFA, 
insulin, glucose enrichment, and D5glycerol enrichment. Starting at time 0, 
each participant will receive a continuous infusion of D5glycerol (0.1 to 0.4 , 
will be generally ~ 0.28 mg/kg Bolus x  over1-5  min (generally around 2 
minutes)   and then 0.01 to 0.04 mg/kg/min will be generally ~ 0.0195 
mg/kg/min)29 and 6,62H2 glucose (initial bolus 3.5 mg/kg * over  1-10 min 
(generally around 7 minutes) , then continuous 0.04 mg/kg/min infusion).30 
Prior to initiation of the insulin clamp (time 120 minutes),  blood will be 
sampled (q5-q10 minutes * 3) for a saved sample of plasma (5 ml) and 
serum (5 ml), glucose, insulin, and glucose enrichment. Next, a 2 hour low-
dose hyperinsulinemic-euglycemic clamp (insulin: 10 mU/m2/min, KPO4 at 
50 ml/hr, 20% Dextrose enriched with 1.5% of 6,62H2 glucose)31,32 will be 
initiated and the dextrose titrated for a target glucose of 90 mg/dl. Serum 
blood glucose will be measured every 10 minutes (Analox, UK) and insulin 
will be measured every 30 minutes. At the end of the 2 hour low-dose 
clamp, a 2 hour high-dose hyperinsulinemic-euglycemic clamp (insulin: 40 
mU/m2/min) will be initiated (time 240 min) with repeat indirect 
calorimetry (440-470 min) and blood sampling will be repeated (q5-q10 
minutes * 3). We may also collect up to 3 urine samples (from the commode 
or urinal) over the course of the visit if the participant happens to void. 

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Anticipated blood obtained during this visit will be about 105 ml (7 
tablespoons). 
  
Visit 6:  This will be similar to Visit 3.  We will collect blood for glucose, 
insulin, HbA1c, lipid, clamp associated analysis and saved blood. We will 
also collect urine at this time if the participant voids.  Anticipated blood 
obtained during this visit will be about 135 ml (9 tablespoons). 
  
Dietary Recall via phone call 
Preparing participants for the dietary recall 
Prior to receiving calls from NCC, the participants will be attending a clinic visit (Visit 1 and 
Visit 5) for the study, at which time they will be prepared for the dietary recall.  The study 
coordinator will do the following: 
• Explain the dietary recall process to the participant by letting them know that: (see 
telephone script) 
o NCC Interviewers will call to ask about everything they had to eat and drink 
the day prior.   
o Participants do not need to keep track of their diet in anticipation of the call.  
Dietary interviewers will help the participant think through their day and 
remember what they had.   
o Calls average around 20-30 minutes.  
• Provide the participant with the Food Amounts Booklet (FAB) that the dietary 
interviewer will refer to during the phone recall. 
• Ask the participant to provide windows of time when they are generally available to 
receive a phone call.  These should not be specific times on specific dates, but 
rather time frames of at least 2-3 hours.  A good example would be “Mondays 
anytime, Tuesdays unavailable, Wednesdays any time after 4pm, etc.…” 
 
Three interviewer-administered 24-hour dietary recalls will be collected from each 
participant during each of measurement periods: 1) Weeks 1-2 of baseline; 2) Weeks 11-12 of 
the intervention (total of 6 recalls/participant). The recalls will be conducted over the 
telephone and will be unannounced (unscheduled within the measurement period) to 
minimize measurement reactivity. The Nutrition Data System for Research (NDSR)33, a dietary 
analysis software program developed and maintained by the University of Minnesota Nutrition 
Coordinating Center (NCC), will be used to collect the dietary recalls.  When collecting recalls 
using NDSR, we will use the multiple-pass interview technique to prompt for complete food 
and beverage recall and descriptions.34 The time of each eating occasion will be also be 
recorded.  A food amount booklet adapted from Van Horn et al35 will be provided to 
participants for use in estimating food and beverage amounts. The dietary recalls will be 
carried out by staff at NCC who are trained and certified in telephone-based collection of 
dietary recalls using NDSR. Interview staff will be blinded to participant intervention 
assignment. NCC has a long history of performing dietary assessment services to researchers. 

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Using 24-hour dietary recall data, average energy intake at each measurement period will 
be calculated for each participant and used to compare pre-post change in energy intake 
between interventions. To evaluate change in diet quality a Healthy Eating Index 2015 (HEI-
2015) score will be estimated for each participant at each measurement period. The HEI-2015 
is a tool developed by the United States Department of Agriculture and the National Cancer 
Institute to evaluate the extent to which diets are consistent with the Dietary Guidelines for 
Americans36,37 Possible total index points range from 0-100, with a higher score indicating 
greater consistency of the diet with the Dietary Guidelines for Americans. Scores for each of 
the 13 components of the index (e.g. total vegetables, whole grains, added sugars) will also be 
calculated. 
 
DXA scan at Laboratory of Integrative Human Physiology (LIHP) 
The Laboratory of Integrative Human Physiology (LIHP) has a Body Composition Testing 
Laboratory which contains equipment for the determination of body composition including 
bone mass, fat mass and lean body mass.  At baseline (visit 2) and 10 weeks (visit 5), all 
subjects will have their height and weight determined using a wall-mounted stadiometer and 
an electronic scale.  Body mass index (BMI) will be calculated as the body weight in kg/height2 
(m2).  Percent body fat and lean muscle mass will be determined by Hologic Horizon A (Hologic 
Inc., Marlborough, MA) dual X-ray absorptiometer (DXA). The DXA will provide bone densities 
and calculations of total body fat, lean body mass and bone mass. The Hologic Horizon A DXA 
can accommodate individuals who weigh up to 500 lbs and will provide calculations of total 
body fat, regional fat, lean body mass, and bone mass. The Hologic Horizon A can provide 
adipose measures of the android and gynoid regions as well as estimation of visceral adipose 
tissue.38 Prior to daily testing the Hologic Horizon A DXA is calibrated using a phantom of 
known composition. The Hologic Horizon A DXA undergoes a yearly preventative diagnostic 
evaluation.  
Lean mass, bone mineral content and fat mass (total, visceral, and 
subcutaneous) will be measured by DXA (Hologic Horizon A) pre and post 
intervention. The Hologic Horizon A will allow regional measurement of trunk, 
android, gynoid, abdominal subcutaneous and abdominal visceral adipose tissue. 
If the Hologic DXA scan is not available, we will use the GE-LUNAR DXA scan 
from Dr. Kelly’s group.  
  
Actigraph Sensor placed at Clinical Research Unit (CRU)  
We will use actigraphy (ie Actigraph Link or Phillips Spectrum) to quantify 
physical activity, sleep duration, and sleep quality. These accelerometry-based 
sensor has been used extensively in clinical research to objectively quantify physical 
activity. The sensors will be worn up to 14 days.  This will be placed at Visit 1 and 
Visit 5. 
  
Glucose Sensor placed at Clinical Research Unit (CRU)  

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We will use a blinded CGM (Freestyle Libre Pro, or DEXCOM G6) to evaluate 
the dietary intervention on the daily glucose profile. The CGM captures interstital 
glucose every 15 minutes to allow characterization of overnight glucose levels, 
average glucose levels and glycemic variability. The sensors will be worn up to 14 
days. It will be placed at Visit 1 and Visit 5. 
 
MRI at Center for Magnetic Resonance Research (CMRR) 
MRI will be used to measure abdominal fat in the liver, visceral fat, and 
ectopic fat in the pancreas and heart 
All MRI scans will be performed on a 3T Prisma MR System (Siemens, 
Erlangen, Germany) located at the UMN Center for Magnetic Resonance Research 
(CMRR) / Center for Clinical Imaging Research (CCIR) at Visit 2 and Visit 5. Hepatic 
fat  (HFF), pancreatic fat, visceral fat, and cardiac fat content will be measured using 
magnetic resonance spectroscopy (MRS).39 Anticipated scan time ~ 1 hour. 
 
Glucose clamp at Clinical Research Unit (CRU) 
For Visit 3 and 6, participants will be provided a fixed meal to eat as dinner the night before 
the study, which they will eat at home. They will be instructed to consume this meal around 5-6 
pm.  They will then remain fasting after eating this meal (water and medications are allowed) 
and will report the next morning around 7 am to the CRU to perform their clamp (Figure 8). At 7 
am the next day, participants will receive D5glycerol and 6,6 2H2 glucose for 2 hours prior to the 
2 step hyperinsulinemic-euglycemic clamp. With this design, baseline lipolysis and endogenous 
glucose production will be measured after a 16 hour fast, which is the same duration as the 
TRE fasting period. 
 
 

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Baseline blood samples will be taken to measure glucose, FFA, insulin, glucose enrichment, 
and D5Glycerol enrichment. Starting at time 0, each participant will receive a continuous 
infusion of D5glycerol  (0.1 to 0.4 , will be generally ~ 0.28 mg/kg Bolus over 1-5 min  
(generally around 2 minutes) and then 0.01 to 0.04 mg/kg/min, will be generally ~ 0.0195 
mg/kg/min)29 and 6,62H2 glucose (initial bolus 3.5 mg/kg over1-10 minute (generally around 7 
minutes), then continuous 0.04 mg/kg/min infusion).30 Prior to initiation of the insulin clamp 
(120 min), indirect calorimetry (80-110 min: ParvoMedics True One 2400 metabolic cart with a 
canopy hood) and blood will be sampled (time 110, 115, 120 min) for resting glucose, insulin, 
and glucose enrichment. Next (120 min), a 3 hour low-dose hyperinsulinemic-euglycemic 
clamp (insulin: 10 mU/m2/min, KPO4 at 50 ml/hr, 20% Dextrose enriched with 1.5% of 6,62H2 
glucose)31,32 will be initiated and the dextrose titrated for a target glucose of 90 mg/dl. Serum 
blood glucose will be measured every 10 minutes (Analox, UK) and insulin will be measured 
every 30 minutes. At the end of the 3 hour low-dose clamp, indirect calorimetry (260-290 min) 
and blood sampling will be repeated (290, 295, 300 min). Finally, a 3 hour high-dose 
hyperinsulinemic-euglycemic clamp (insulin: 40 mU/m2/min) will be initiated (300 min) with 
repeat indirect calorimetry (440-470 min) and blood sampling will be repeated (470, 475, 480 
min). To account for the tracer enrichment in the dextrose solution during the insulin clamp, 
equations described by Finegood40 will be used to calculate Ra and Rd.  Hepatic glucose 
output, hepatic insulin sensitivity, oxidative glucose disposal, and nonoxidative glucose 
disposal will be calculated similar to published literature.41,42 Metabolic flexibility will be 
calculated as RQclamp-RQ fast.43,44 Glucose enrichment will be measured by Dr. Bryan Bergman’s 
laboratory; Dr Bergman directs the Molecular and Cellular Analytical Core Facility as part of the 
Colorado NORC (P30DK48520) and has an extensive history performing stable isotopes 
analyses.42,45 
A key feature of reduced metabolic flexibility is impaired suppression of lipolysis in 
response to insulin.46 After the evening meal, the participant will remain fasting until study 
conclusion. A continuous infusion of D5Glycerol will be started 2 hours before the clamp and 
continued through the clamp. Blood samples will be collected for plasma D5 glycerol to 
calculate lipolysis. This will be measured at baseline (16 hours from the last meal, 2 hours after 
starting the D5Glycerol infusion) (Fig 8), at the end of the low-dose clamp and at the end of 
the high-dose clamp. Calculation of lipolysis will be performed as previously described.29 As 
another measure of adipose tissue sensitivity, we will also examine the difference between 
FFAclamp vs FFAfast. Dr Bergman’s laboratory will measure the FFA concentration and 
enrichment. 
We will also collect up to 3 urine samples (from the commode or urinal) over the course of 
the visit when the participant happens to void, or use the restroom. 
Blood will be drawn here for stable isotope analysis.  The total amount of blood taken will 
be about 8-10 tablespoons during the course of the clamp. 
  
Indirect calorimetry at Clinical Research Unit (CRU)  

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This will be performed during the CRU stay.  This test will be done to look 
at the oxygen and carbon dioxide from the expired breath. The participant will lie down in 
a bed at the CRU with a special facemask for 30 minutes at rest. We will then capture the 
expired oxygen and carbon dioxide for another 30 minutes. This will be repeated 2 more 
times during the clamp, for a total of 3 times with each clamp.   
  
Aim Specific Outcome Measures are noted in the respective sections below 
Aim#1: Evaluate the effect of TRE with ad libitum intake on weight and body composition 
Rationale: TRE is associated with prolonged daily fasting. Prolonged fasting mobilizes adipose 
tissue triglycerides, with 60% of the increase in lipid kinetics occurring between 12-24 hours of 
fasting.24 Although we reported that TRE reduces weight compared with non-TRE (Fig 2) it 
remains unknown:1) How TRE weight-loss may compare with CR weight loss, 2) Whether 
weight loss associated TRE (prolonged daily fasting) or CR (reduced caloric intake over a 
prolonged eating window) may differentially alter body composition.  
 
Hypothesis : H 1.1: The TRE and CR groups will have similar weight loss, which will be greater 
than weight loss achieved in the non-TRE group (primary outcome). H 1.2: TRE will result in 
greater loss of loss of total body fat (quantified by DXA) and greater loss of hepatic/visceral 
fat/ectopic fat (quantified by MRI) than CR. 
 
Outcome measure: Weight and body composition [Dual X-ray absorptiometry (DXA)]  
Weight (primary outcome) will be measured at baseline, Week 6 and Week 12. Lean mass, 
bone mineral content and fat mass (total, visceral, and subcutaneous) will be measured by 
DXA (Hologic Horizon A) pre and post intervention. The Hologic Horizon A will allow regional 
measurement of trunk, android, gynoid, abdominal subcutaneous and abdominal visceral 
adipose tissue.38,47 
 
Outcome measure: Magnetic Resonance Imaging - Liver Fat  
Measurement of Liver and Pancreatic Fat Content via MRI 
Each participant will have Hepatic Fat Fraction (HFF) measured by MRI via 1H- magnetic 
resonance spectroscopy (MRS) at the Magnetic Resonance Research Institute at the University 
of Minnesota. HFF will be measured with single-voxel 1H-MRS on a 3.0 T Prisma whole body 
MRI scanner (Siemens Medical, Erlangen, Germany) using the software package provided by 
the vendor (the HISTO sequence from Siemens’ LiverLab). After localizer and anatomical 
images, a single voxel (3x3x3 cm) will be positioned in the right lobe of the liver while avoiding 
hepatic and portal vessels. A single volume stimulated echo acquisition mode (STEAM) 
measurement (TR/TM = 3000/10 ms) without water or lipid suppression will be acquired at 5 
TE values (TE=12, 24, 36, 48, 72 ms) over a single breath-hold to enable measurement and 
correction of T2 effects. The HISTO program fits the water and lipid peaks and calculate a liver 
fat percentage. The MRS measurement will be performed 2 times to assess repeatability of 
HFF concentration. In our preliminary work, the coefficient of variation between 

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measurements was 0.78% with an inter-class correlation coefficient of 0.936, showing high 
repeatability and reproducibility. The pancreatic fat fraction will be measured using a similar 
MRS method, but with a smaller voxel size (2x2x2 cm) and two averages to improve signal-to-
noise. Additionally, a proton density fat fraction image will be acquired using a chemical-shift 
encoded (CSE-MRI) method (LiverLab’s “qdixon”, 3D gradient echo, TR=9ms, flip angle=4°, 
TE=1.15, 2.46, 3.69, 4.92, 6.15, 7.38 ms, 2x2x3.5mm resolution, one 17s breath-hold) to 
evaluate spatial distribution of fat in both liver and pancreas. The MRS measurements of 
hepatic and pancreatic fat will be used as the primary metric. CSE-MRI has shown promise as a 
measure of steatosis staging compared to liver biopsy.48  A HFF of ≥ 5.5% will be used to 
diagnose NAFLD, which has been previously shown to be sensitive and specific for detecting 
NAFLD in adults49 and in children versus liver biopsy.50  
 
 
Outcome Measure: Magnetic Resonance Imaging – Visceral Fat 
In addition to DXA, Visceral fat will also be measured by MRI.51,52 The volume of visceral 
adipose tissue will also be measured using a CSE-MRI method optimized for full abdominal 
coverage.15 The CSE-MRI acquisition (qdixon, sagittal 3D gradient echo, TR=9ms, flip angle=4°, 
TE=1.15, 2.46, 3.69, 4.92, 6.15, 7.38 ms, 3.2x3.2x3 mm resolution, one 22s breath-hold) will 
produce a 3D isotropic fat fraction map covering from the liver dome to pelvic floor. The images 
will be reformatted in the axial plan and processed using ITK-SNAP.16 The fat fraction image will 
be automatically thresholded to remove signal from background and air cavities. Manual 
processing will be used to exclude arms and breast tissue, and to delineate the visceral, 
subcutaneous, and vertebral/intramuscular compartments. From these the visceral (VAT), 
subcutaneous (SAT), and total adipose tissue (TAT) volumes (mL) will be extracted.  
 
Exploratory Outcome: Magnetic Resonance Imaging - Ectopic fat in the pancreas and heart 
Pancreatic fat quantification 
The pancreatic fat fraction will be measured using a similar MRS method, but with a 
smaller voxel size (2x2x2 cm) and two averages to improve signal-to-noise. Additionally, a 
proton density fat fraction image will be acquired using a chemical-shift encoded (CSE-MRI) 
method (LiverLab’s “qdixon”, 3D gradient echo, TR=9ms, flip angle=4°, TE=1.15, 2.46, 3.69, 
4.92, 6.15, 7.38 ms, 2x2x3.5mm resolution, one 17s breath-hold) to evaluate spatial 
distribution of fat in both liver and pancreas. The MRS measurements of hepatic and 
pancreatic fat will be used as the primary metric. CSE-MRI has shown promise as a measure of 
steatosis staging compared to liver biopsy.48  A HFF of ≥ 5.5% will be used to diagnose NAFLD, 
which has been previously shown to be sensitive and specific for detecting NAFLD in adults49 
and in children versus liver biopsy.50 
Cardiac (Ectopic) Fat Quantification via MRI 
1H magnetic resonance spectroscopy (MRS) is non-invasive in vivo measurement of 
steatosis applied in youth.45It permits precise and reproducible quantitation of intracellular 
triglyceride content in the cytosol of non-adipose cells. As previously described, a 
spectroscopic volume of interest (a single voxel, 0.8×1.8×2.4cm3) will be positioned over the 

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interventricular septum using end-systolic cardiac cine images in two cardiac planes (short axis 
and semi 4 chamber axis), collected at end-expiration.53 During acquisition, participants 
breathe freely, with data acquisition triggered simultaneously at end systole (via ECG gating) 
and end expiration (via a respiratory navigator PACE). Spectra are collected without water 
suppression using the following parameters: repetition time was ∼4s depending on HR; echo 
time, 35ms; 1,024 data points over a 2,000-Hz spectral width; and 32 acquisitions. 
Spectroscopy data will be processed by Dr. Bolan using commercial software (NUTS, Acorn 
NMR, Fremont, CA) as previously described.49 Final calculation of fat and water signal 
intensities will account for fat and water signal decay due to spin-spin relaxation.49,54 
Myocardial TG will be expressed as a percentage of tissue water content. Imaging extracellular 
fat (pericardial fat) content will be performed using a Turbo FLASH sequence with a multi-slice 
2D transverse acquisition and single-shot per slice, as previously described.55  We will acquire 
13 slices in total, 6-mm thickness with 6-mm slice gap, FOV=400×400mm2, matrix=144×192, 
spatial resolution=2.78×2.08×6mm3, flip angle=10, TR/TE=3.2/1.27ms, receiver 
bandwidth = 651 Hz/pixel. Pericardial fat quantification will be performed using commercially-
available ORS Visual Imaging Software (Object Research Systems, Inc., Montreal, Canada), in 
the following ways: 1) as volume (cm3) in a 6 mm slab at the level of the left main origin; 2) as 
volume (cm3) in a 6 mm slab at the level of the right ventricular (RV) free wall; and 3) as 
thickness (mm) of the pericardial fat at RV free wall in the same slice Tracing of the fat border 
will be manually performed by an experienced reader, blinded to all participant characteristics. 
 
 Aim#2: Assess the effect of TRE with ad libitum intake on caloric balance. 
Rationale: By prolonging the fasting period, TRE reduces the available eating window and 
therefore may reduce caloric intake. This was suggested by previous literature [estimated 
energy intake declined by 341±53 kcal/day]6 and our preliminary data.It remains unknown 
whether reducing the eating window may alter nutritional density of ingested foods or alters 
other non-eating behaviors in compensation. 
Hypothesis: H 2.1:TRE reduces caloric intake [gold-standard interviewer administered 24-hour 
dietary recall compared with non-TRE and similar to CR, H.2.2: TRE will not alter physical 
activity and will increase fat oxidation compared with CR or non-TRE. 
 
Outcome Measure: Caloric intake as measured by 24-hour dietary recall (primary outcome) 
Three interviewer-administered 24-hour dietary recalls will be collected from each 
participant during each of measurement periods: 1) Weeks 1-2 of baseline; and 2) Weeks 11-
12 of the intervention (total of 6 recalls/participant). The recalls will be conducted over the 
telephone and will be unannounced (unscheduled within the measurement period) to 
minimize measurement reactivity. The Nutrition Data System for Research (NDSR)33, a dietary 
analysis software program developed and maintained by the University of Minnesota Nutrition 
Coordinating Center (NCC), will be used to collect the dietary recalls.  When collecting recalls 
using NDSR, we will use the multiple-pass interview technique to prompt for complete food 
and beverage recall and descriptions.34 The time of each eating occasion will be also be 

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recorded.  A food amount booklet adapted from Van Horn et al35 will be provided to 
participants for use in estimating food and beverage amounts. The dietary recalls will be 
carried out by staff at NCC who are trained and certified in telephone-based collection of 
dietary recalls using NDSR. Interview staff will be blinded to participant intervention 
assignment. NCC has a long history of performing dietary assessment services to researchers. 
Using 24-hour dietary recall data, average energy intake at each measurement period will 
be calculated for each participant and used to compare pre-post change in energy intake 
between interventions. To evaluate change in diet quality a Healthy Eating Index 2015 (HEI-
2015) score will be estimated for each participant at each measurement period. The HEI-2015 
is a tool developed by the United States Department of Agriculture and the National Cancer 
Institute to evaluate the extent to which diets are consistent with the Dietary Guidelines for 
Americans36,37 Possible total index points range from 0-100, with a higher score indicating 
greater consistency of the diet with the Dietary Guidelines for Americans. Scores for each of 
the 13 components of the index (e.g. total vegetables, whole grains, added sugars) will also be 
calculated. 
 
 
Outcome Measures: Fat oxidation/Basal Metabolic rate 
This will be measured by indirect calorimetry (Described in Aim 3). 
 
Outcome Measures: Physical activity.  
All participants will wear the Actigraph Link (up to 2 weeks) pre and post-intervention  
 
Aim#3: Determine the impact of TRE with ad libitum intake on metabolic flexibility 
Rationale: Metabolic flexibility is the adaptability of fuel selection given ambient fuel 
exposure. Adverse health, such as obesity, insulin resistance, type 2 diabetes and sedentary 
behavior, are associated with metabolic inflexibility.56,57 In contrast, exercise and CR induced 
weight loss enhances metabolic flexibility.57 In animals, restricting the eating window improves 
metabolic flexibility by enhancing fat oxidation while fasting and augmenting carbohydrate 
oxidation while feeding.1 In humans, higher fat oxidation stabilizes weight gain in response to 
chronic overfeeding. 58 A longitudinal study in Pima Indians reported that participants who 
were low fat oxidizers [90%tile of 24-hr RQ] were at 2.5 times higher risk of gaining ≥5 kg body 
weight (25 months follow up±11 months) than those who were high fat oxidizers [10%tile of 
24-hr RQ].59 Similar findings have been observed in Caucasian males; non-obese men who are 
low fat oxidizers (fasting RQ ≥ 0.85) were 2.42 times (95% CI 1.10-5.32) more likely to gain ≥5 
kg over 10 years than non-obese men who are high fat oxidizers (fasting RER < 0.76).60 This 
Aim has the following conceptual model (Fig 1): TRE results in daily prolonged fasting. Chronic 
daily prolonged fasting enhances metabolic flexibility, with enhanced lipolysis and fat 
oxidation while fasting and enhanced glucose oxidation with glucose exposure (i.e. 
hyperinsulinemic-euglycemic clamp setting). Augmenting metabolic flexibility will reduce 
systemic fat depots (Aim 1) and improve insulin sensitivity (Aim 3).  

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Hypothesis: H 3.1: TRE will enhance metabolic flexibility compared with CR and non-TRE as 
measured by indirect calorimetry at rest [before and during 2 step 6,6-2H2 hyperinsulinemic-
euglycemic clamp: primary outcome.H 3.2: TRE will improve insulin sensitivity compared with 
non-TRE and similar to CR. H 3.3: TRE will augment fasting lipolysis as measured by D5Glycerol 
and enhance lipolysis  
 
Outcome Measure: Metabolic flexibility – RQ response to hyperinsulinemic-euglycemic 
clamp  
Each participant will receive a standardized mixed meal at 5 pm to take home. After the 
mixed meal, the participant will fast (water permitted) overnight (Fig 8). At 7 am the next day, 
participants will receive D5Glycerol and 6,6 2H2 glucose for 2 hours prior to the 2 step 
hyperinsulinemic-euglycemic clamp. With this design, baseline lipolysis and endogenous 
glucose production will be measured after a 16 hour fast.  
Baseline blood samples will be taken to measure glucose, FFA, insulin, glucose enrichment, 
and glycerol enrichment. Starting at time 0, each participant will receive a continuous infusion 
of D5glycerol  (0.1 to 0.4 , will be generally ~ 0.28 mg/kg Bolus over 1-5 min (generally around 
2 minutes)  and then 0.01 to 0.04 mg/kg/min, will be generally ~ 0.0195 mg/kg/min)29 and 
6,62H2 glucose (initial bolus 3.5 mg/kg *over 1-10 minutes (generally around 7 minutes) , then 
continuous 0.04mg/kg /min infusion).30 Prior to initiation of the insulin clamp (120 min), 
indirect calorimetry (80-110 min: ParvoMedics True One 2400 metabolic cart with a canopy 
hood) and blood will be sampled (time 110, 115, 120 min) for resting glucose, insulin, and 
glucose enrichment. Next (120 min), a 3 hour low-dose hyperinsulinemic-euglycemic clamp 
(insulin: 10 mU/m2/min, KPO4 at 50 ml/hr, 20% Dextrose enriched with 1.5% of 6,62H2 
glucose)31,32 will be initiated and the dextrose titrated for a target glucose of 90 mg/dl. Serum 
blood glucose will be measured every 10 minutes (Analox, UK) and insulin will be measured 
every 30 minutes. At the end of the 2 hour low-dose clamp, indirect calorimetry (200-230 min) 
and blood sampling will be repeated (230,235,240 min). Finally, a 2 hour high-dose 
hyperinsulinemic-euglycemic clamp (insulin: 40 mU/m2/min) will be initiated (240 min) with 
repeat indirect calorimetry (320-350 min) and blood sampling will be repeated (350,355,360 
min). To account for the tracer enrichment in the dextrose solution during the insulin clamp, 
equations described by Finegood40 will be used to calculate Ra and Rd.  Hepatic glucose 
output, hepatic insulin sensitivity, oxidative glucose disposal, and nonoxidative glucose 
disposal will be calculated similar to published literature.41,42 Metabolic flexibility will be 
calculated as RQclamp-RQ fast.43,44 Glucose enrichment will be measured by Dr. Bryan Bergman’s 
laboratory; Dr Bergman directs the Molecular and Cellular Analytical Core Facility as part of the 
Colorado NORC (P30DK48520) and has an extensive history performing stable isotopes 
work.42,45 
 
Outcome Measure: Lipolysis alterations in response to hyperinsulinemic-euglycemic clamp 
A key feature of reduced metabolic flexibility is impaired suppression of lipolysis in response to 
insulin.46 After the evening meal, the participant will remain fasting until study conclusion. A 
continuous infusion of albumin-bound D5Glycerol, will be started 2 hours before the clamp 

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and continued through the clamp. Blood samples will be collected for plasma D5Glycerol to 
calculate lipolysis. This will be measured at baseline (16 hours from the last meal, 2 hours after 
starting the D5Glycerol infusion) (Fig 8), at the end of the low-dose clamp and at the end of 
the high-dose clamp. Calculation of lipolysis will be performed as previously described.29 As 
another measure of adipose tissue sensitivity, we will also examine the difference between 
FFAclamp vs FFAfast.Dr Bergman’s laboratory will measure the FFA concentration and 
enrichment. 
 
 
 

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Schedule of Events 
 
Visit 0 
(Virtual) 
Visit 1 
(In Person-
CRU) 
Phone Call 
Visits 1 
Visit 2 
(In Person –
Mariucci & 
CMRR) 
Visit 3 
(In Person-CRU) 
Meal picked up 
night before 
Visit 4 
(Virtual) 
 
Visit 5 
(In Person –
Mariucci & 
CMRR) 
Phone Call 
Visits 2 
Visit 6 
(In Person-
CRU) 
Meal picked up 
night before 
 
Screening 
Baseline Visit 
1 
3 Dietary 
Recall Phone 
Calls 
Baseline Visit 
2 
Baseline Visit 3 
(Randomization) 
Mid 
Intervention 
Visit 
End Study Visit 
1 
3 Dietary Recall 
Phone Calls 
End Study Visit 
2 
 
Pre 
Intervention 
Pre 
Intervention 
Pre 
Intervention 
Pre 
Intervention 
Week 1 of 
Intervention 
Week 6 of 
Intervention 
Week 10 of 
Intervention 
Weeks 11&12 
of Intervention 
Week 12 of 
Intervention 
Time Point 
10-14 days 
prior to Visit 1 
Day 1 
2 week period 
between Visits 
1 & 2 
2-3 weeks 
after Visit 1 
1-7 days after 
Visit 2 
6 weeks 
after Visit 3 
4 weeks after 
Visit 4 
2 week period 
between Visits 
5 & 6 
2-3 weeks after 
Visit 5 
Participant Stipend 
$10 
$ 15  
$25 
$50 
$100 
$25  
$50 
$25  
$100  
Consent 
X 
 
 
 
 
 
 
 
 
Survey 
X (eating 
disorder) 
X  (appetite 
sensation) 
 
 
X (SF-36) 
X  (appetite 
sensation)  
X  (appetite 
sensation) 
 
X (SF-36) 
Height/Weight 
X (self-report) 
X  
 
X 
X 
X (self-
report) 
X  
 
X 
mCC app 
downloaded 
X  
 
 
 
 
 
 
 
 
Ongoing mCC 
monitoring 
 
X 
X 
X 
X 
X 
X 
X 
X 
Blood draw (fasting) 
 
X1,X2 
 
 
X3 
 
 
 
X1,3,4 
Urine sample 
 
 
 
 
X 
 
 
 
X 
Dietary recall 
 
 
X 
 
 
 
 
X 
 
DEXA Scan (fasting) 
 
 
 
X 
 
 
X 
 
 
Wearable sensor 
distributed (CGM) 
 
X 
 
 
 
 
X (after MRI) 
 
 
Wearable sensor 
removed (CGM) 
 
 
 
X (before MRI) 
 
 
 
 
X  
Wearable sensor 
distributed 
(Actigraph) 
 
X 
 
 
 
 
X (after MRI) 
 
 
Wearable sensor 
removed(Actigraph) 
 
 
 
X (before MRI) 
 
 
 
 
X  
MRI (1 hour scan) 
 
 
 
X 
 
 
X  
 
 
Dietician counseling: Initial dietary telephone counseling to all 3 groups after Visit 3. Dietician to call TRE and CR groups for dietary counseling weekly for 1st 4 weeks and every 2 weeks 
thereafter. 
mCC logging feedback: Weekly email/text (participant preference) on mCC logging to all groups starting after Visit 1. 

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Inpatient CRU stay 
for clamp/fat 
breakdown 
measurements 
(7am) 
 
 
 
 
X 
 
 
 
X 
Indirect calorimetry 
 
 
 
 
X 
 
 
 
X 
 
X1 Fasting glucose, 
insulin, whole blood for 
saved serum & plasma 
 
 
 
 
 
 
 
 
 
X2 CMP, HbA1c, TSH, 
Hgb, Lipid 
 
 
 
 
 
 
 
 
 
X3 Clamp procedure, 
saved samples of 
serum, plasma, and 
whole blood 
 
 
 
 
 
 
 
 
 
X4 HbA1c, Lipid 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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5.3 
Study Duration: The anticipated duration for  
● 
Each subject (screening visit to study completion): 6 months 
● 
Screening of subjects (4 years) 
● 
Completion of study visits (4 years)  
 
 
5.4 Use of radiation: Yes, we will use DXA to measure body composition before and 
after the intervention.    
5.5 Use of Center for Magnetic Resonance Research: Yes, we will use CMRR to 
measure ectopic fat by 3T MRI before and after the intervention. The research 
team will utilize the CMRR Center’s screening tools and adhere to the screening 
SOP during enrollment of all research participants in this protocol.  The CMRR 
Center’s screening tools and SOP are IRB approved under the CMRR Center Grant 
(HSC# 1406M51205) and information regarding screening procedures is publically 
available on the CMRR website (CMRR Policies / Procedures). 
6.0 
Data and Specimen Banking 
6.1 
Storage and Access: The data will be stored electronically in the CTSI REDCap 
database as well as a secured AHC server designated for research purposes. Access to 
these databases will be through AHC computers, which are password protected in a 
locked office area. Hard copies of the data will be stored in a locked cabinet in a 
secured area in Endocrinology. De-identified data will be uploaded to the cloud 
(smartphone app data, wearables data, and CGMS data) and then downloaded by 
study staff. After the study is completed, the de-identified, archived data will be 
transmitted to and stored at the Salk Institute and University of Minnesota, for use by 
other researchers including those outside of the study.  
Paper data will be stored in the locked office of the PI. REDCap is an electronic system 
that is accessed via a secure web interface. 
The data will be stored electronically in the CTSI REDCap database as well as a 
secured AHC server designated for research purposes. Access to these databases 
will be through AHC computers which are password protected in a locked office 
area. Hard copies of the data will be stored in a locked cabinet in a secured area 
in Endocrinology. 
 
With the participant’s approval and as approved by the UMN IRB, de-identified 
biological samples will be stored at Dr. Mashek’s laboratory or AHC Biorepository.  

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The samples will be stored using a code-link that will allow linking the biological 
specimens with the clinical/phenotypic data from each participant, maintaining 
the masking of the identity of the participant. The sample and associated data will 
be stored indefinitely. Only authorized study/repository staff will have access to 
the specimens and data. Genetic testing will not be performed.  
During the conduct of the study, an individual participant can choose to withdraw 
consent to have biological specimens stored for future research. However, 
withdrawal of consent with regard to biosample storage will not be possible after 
the study is completed. 
 
The samples may also be analyzed by the study team’s collaborators (internal or 
external) for additional analysis. If the samples are sent externally, a material 
transfer agreement will be signed. All samples will be sent deidentified. The 
samples may be analyzed for various metabolites/chemicals but will not be 
analyzed for genetic testing.  
 
 
6.2 Data:  
• Phone screening: Screening interview with personal information 
o This will include name, birthdate, email address, phone number, 
home address, and medical record number 
• Visit 1-6: History and Measurement data. This will include information 
about glucose profiles( up to 14 days), physical activity, body composition 
(By DXA and MRI), plasma samples, details about dietary intake (24 hour 
recall) 
• Dietary program: Documentation of compliance with dietary program 
through the MyCircadianClock application 
6.3 Release/Sharing: 
We attest that we will register this clinical trial in ClinicalTrials.gov prior to any 
subject enrollment. Applicable results will be posted upon completion of the project 
and specified timeline as required by the NIH policy on the dissemination of NIH-
funded clinical trials information. The informed consent documents for this study will 
include a specific statement regarding the posting of the clinical trial information at 
ClinicalTrials.gov.  The University of Minnesota has an institutional policy ensuring that 
clinical trials registration and results reporting occur in compliance with policy 
requirements.  
This data will also be available for future use by other investigators if proper IRB 
approval and material transfer agreements are obtained. The data will be made 
available after the main findings from the final research data set have been accepted 
for publication. This shared data will be provided de-identified for analysis. 

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7.0 
Sharing of Results with Participants 
7.1 
Each participant’s individual results will be provided to the specific individual. 
The results will otherwise be analyzed using de-identified data for presentation at 
national meetings and publication in relevant journals 
7.2 
Sharing of genetic testing: N/A 
 
8.0 
Study Population 
8.1 Inclusion Criteria:  
• Age 18-65, 
• BMI ≥30 and ≤ 55 kg/m2 
• Own a smartphone compatible with the myCircadianClock phone 
application.  
• Self-reported habitual wakening between 5-9 am,  
• Self reported sleep duration of 6-9 hours 
• Weight must be stable [+/- 5 pounds] for at least 3 months prior to the 
study. 
• Eating window (time between 1st food intake and last food take) ≥12 
hours using mCC 
• Able to understanding English 
8.2 Exclusion Criteria:  
• Beta blockers or medications known to affect weight such as TZD, insulin, 
GLP-1 agonists, phentermine, or sibutamine. 
• Shift work (working from 11pm to 7 am) 
• Clinically significant medical issues (diabetes, cardiovascular disease, 
uncontrolled pulmonary disease),  
• A history of abnormal laboratory results, such as hematologic (platelets < 
100), hepatic (LFTs > 2X nl), renal (Cr > 1.5) 
• MRI contraindication (metal in body, claustrophobia). 
• Eating window < 12 hours 
• Unable to consistently document food intake using the myCC app (need at 
least 2 eating occasions>  6 hours apart on a given day for at least 50% of 
days) 
• Pregnancy and illiteracy 
• Concern for active eating disorder per screening questionnaire 
• Self-reported eating disorder or history of eating disorder 
8.3 Screening:  

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The screening phone call will introduce the study (see phone script), review study 
eligibility (including a 5 question survey to screen for disordered eating) and 
include a verbal consent for fasting.27 
We will request a waiver of documentation of consent during the phone 
screening.   
The Screening Visit (Visit 0) will be a virtual visit using Zoom or Doximity. 
Informed consent will be obtained by the study team. The entire consent 
document will be reviewed, including all study procedures and expectations, 
risks, benefits, and what volunteering means. Candidates will be given time to 
read the consent, ask questions, and to take the consent home to review if 
requested. Height and weight will be self-reported. An eConsent/HIPAA form will 
be developed in REDCap and provided to potential participants for esignature. 
Paper copies will be used dependent on participant preference. 
 
 
The current UMN consent/HIPAA form will include Salk approved consent 
language for the mCC app usage. The signed consent/HIPAA form will be 
applicable for both UMN and Salk procedures. Once the participant has signed 
the consent/HIPAA form, the mCC app will be downloaded from the iTune 
Appstore or Google Play appstore. After activation of the app and device ID 
authentication, the study code cannot be concurrently used with another device. 
A unique random alphanumeric character string will be used to encode the user 
and only that identifier will be used to transmit encrypted data between the 
device and a HIPAA compliant cloud server.  
 
Once consented using the site consent form and the online app consent form, the 
subject will be scheduled for their baseline appointment (Visit 1).  
 
 
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 

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participation in the study.  
Children greater than age 18 will 
be allowed to participate. 
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 
Included/Allowed to Participate 
Active members of the military 
(service members), DoD personnel 
(including civilian employees) 
Included/Allowed to Participate 
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. 
Included/Allowed to Participate 

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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:  
Undervalued/disenfranchised/Active members of the military and DoD 
employees will not be identified and information will not be collected as to a 
patient’s status. This research does not add risk to this group. We will not 
specifically recruit from this population but will not exclude a participant from 
this population if they volunteer and are eligible for the study.  
 
10.0 Local Number of Participants 
10.1 Local Number of Participants to be Consented: We anticipate screening and 
consenting ~250 people in person. We anticipate recruiting ~ 90 participants for 
which 72 will complete the study.  
11.0 Local Recruitment Methods 
11.1 Recruitment Process: The success in recruitment lies in a multimedia approach, 
including recruitment through the electronic health record, online advertisement, 
newspaper, television and radio public interest stories; public service 
announcements; solicitation of health care provider referrals; direct mailings to 
potential participants; advertisements; small print media, registration in 
StudyFinder and Clinicaltrials.gov and creation of a study website similar to 
previous clinical trials at the University of Minnesota. Based on our success with 
our current R01 recruitment, we will rely heavily on using the electronic health 
record (EHR:EPIC) for recruitment (BPIC based) 
Potentially, individuals who are a patient of the Endocrinology Clinic and have an 
upcoming visit will be approached at that visit. Otherwise, we may also have their 
primary endocrinologist sign a letter supporting their eligibility which we will mail 

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to the patients and then follow up with a phone call. We will only call participants 
who are part of the group practice for the Endocrinology division to follow up on 
the mailed letters.  
If the individual is not part of the Endocrinology clinic, the general recruitment 
will use BPIC to identify the patients and Fairview Research Services/Addressing 
and Mailing to mail study information to potentially eligible patients explaining 
the study and asking them to contact research staff. 
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. It was created through the 
Clinical & Translational Science Awards Consortium in 2009. 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. 
11.2 Identification of Potential Participants: The University of Minnesota is a partner of 
the Fairview Health System which has used EPIC since 2011. As of January 2019, 
the Fairview EHR has ~2.8 million living patients. Of these patients, 1,700 had 
prediabetes diagnosed either as fasting glucose between 100-125 (inclusive) or 
HbA1c between 5.7-6.4 (inclusive)  within the last 3 months (Data from February 
2019 – May 2019) and have given consent for participation. Using the recruitment 
infrastructure in place with R01, we anticipate a 10% response rate (n=170) every 
3 months. BPIC will identify potential participants by applying the eligibility 
criteria for the study to the EHR record. The first screen will encompass the most 
recent 6 months review of the EHR. Subsequent screens will include only the past 
month review of eligible participants. Patients who have opted out of research in 
their Epic EHR will be excluded from consideration for participation.  
11.3 Recruitment Materials:  
Participants may self-refer if they become aware of the study through 
recruitment materials or word of mouth referrals. [A letter about the study may 
be sent to potential participants, asking them to contact the study coordinator if 
they are interested.] Individuals who respond to the recruitment materials will be 
taken through a phone-screening questionnaire. If following the phone screen the 
potential participant is eligible and interested, the study coordinator will schedule 
the screening study visit. 
The study will utilize social media accounts such as (Facebook, Twitter, Instagram, 
independent website, etc.) to create an informative space about research 
opportunities and information for patients with obesity or prediabetes.  
11.4 Strategies to be implemented in the event of enrollment shortfalls: 

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Recruitment rates will be reviewed monthly. If recruitment is not proceeding as 
planned, we will recruit from additional health plans in the Twin Cities that have 
previously worked with the University of Minnesota on clinical trials, including 
Hennepin County Medical Center, Health Partners, and the Allina Health System. 
These health systems also have electronic health records in place to target 
recruitment to potentially eligible participants. We will also engage electronic 
media to raise the study profile within the community, including television and 
radio. Other options will include online advertisement, newspaper, television and 
radio public interest stories; public service announcements; solicitation of health 
care provider referrals; direct mailings to potential participants; advertisements; 
small print media, registration in StudyFinder and Clinicaltrials.gov and creation of 
a study website similar to previous clinical trials at the University of Minnesota. 
11.5 Participant stipend: Subject compensation will be tied with retention. For the 
screening visit, the subjects will be paid $25. If they are eligible for the study and 
complete the baseline visits (Visit 3), they will be paid $125. The will receive $50 
for the midpoint visit (Visit 4) and $200 for completing the entire study (Visit 7). If 
the subject successfully completes the entire study, monetary compensation will 
be $400.  Additional retention techniques will include the following: 1) Upfront 
notice during phone screening and screening visit about time needed for study 
commitment, 2) Prorated compensation, 3) Tailoring dietary interventions to 
improve palatability while remaining within the parameters. Compensation will 
be provided by a check mailed to the participant. To increase retention in the 
non-TRE (control) group, we will offer a complimentary dietary session after the 
intervention and Visit 6 have been completed to point out dietary changes the 
participant may wish to consider to support good nutrition and health body 
weight. A resource book may also be provided. 
12.0 Withdrawal of Participants 
12.1 Withdrawal Circumstances:  
• Stopping rules met 
• Participant request 
• Per judgement of PI 
12.2 Withdrawal Procedures: If the participants want to discontinue their dietary 
intervention, we will offer the following options in a sequential fashion: 1) 
Address any limiting factors that may prevent program completion, 2) Move the 
final assessment sooner to be performed at the time of dropping out, 3) Continue 
data collection as intensively as possible given the participant’s preferences. We 
will also collect reasons for drop out (i.e. time/duration, transportation, side 
effects, perceptions of efficacy, interaction with staff) to use as potential 
covariates in the statistical model.  In terms of missing data, results from the last 
observation carried forward as well as multiple imputation analysis61 For the final 
analysis, intention to treat analysis and per-protocol analysis (analysis in those 
who completed the protocol) will be performed. 

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12.3 Termination Procedures: This will be the same as the withdrawal procedures as 
previous described. 
13.0 Risks to Participants 
13.1 Foreseeable Risks: Foreseeable Risks:  
There will be potential physical risks associated with the study as described in 
next section. The seriousness of these risks is low. The risks to the subjects arise 
from using established laboratory techniques (Venipuncture, hyperinsulinemic, 
euglycemic clamp, stable isotope tracers, DXA, CGMs, MRI). The infusions used in 
the study are naturally occurring (i.e. insulin, glucose, potassium, D5Glycerol, 6,6 
2H2 glucose ) and have a history of being successfully used previously in human 
studies.  
 
All adverse events (AEs) must have their relationship to study intervention 
assessed by the clinician who examines and evaluates the participant based on 
temporal relationship and his/her clinical judgment. The degree of certainty 
about causality will be graded using the categories below. In a clinical trial, the 
study product must always be suspect. 
 
• Related – The AE is known to occur with the study intervention, there is a 
reasonable possibility that the study intervention caused the AE, or there is a 
temporal relationship between the study intervention and event. Reasonable 
possibility means that there is evidence to suggest a causal relationship between 
the study intervention and the AE. 
• Not Related – There is not a reasonable possibility that the administration of 
the study intervention caused the event, there is no temporal relationship 
between the study intervention and event onset, or an alternate etiology has 
been established 
 
 
Study Procedure 
Risk 
Likelihood/seriousness/mitigation 
Phone screening 
Breach of confidentiality 
Low/Low. We will ask questions related to health, 
fitness, and eligibility for the study. Data will be 
stored on password protected computer in locked 
area. 
Periods of fasting 
Hunger, lightheadedness 
Low/Low. This will be kept to less than 24 hrs. 
Adequate fluids (ie water) will be given to minimize 
dehydration. 

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History data 
Incidental findings, breach of 
confidentially 
Low/Low. We will exclude subjects if history is 
clinically relevant. 
Venipuncture lab 
tests 
Incidental findings,  
Transient 
pain/bleeding/bruising; 
High/Low. This is a standard blood draw and total 
amount of withdrawn blood will be held to IRB 
guidelines. 
MRI 
Claustrophobia, 
disruption/heating of  metal 
implants  
Low/Low. Screening questionnaire will be performed 
prior to at the phone screen and repeated just prior 
to MRI. Any participants who have a contraindication 
to MRI will be excluded from imaging. 
DXA 
Radiation exposure 
High/Low. This is less than one day from the natural 
background radiation in Minnesota. 
CGM 
Rash on Skin, irritation to 
skin from adhesive pads, 
incidental findings 
Low/Low. The CGM  results will be blinded to the 
participant. The goal is to monitor glycemic 
fluctuations with exercise. If significant 
hyperglycemia or hypoglycemia is noted after 
unblinding, the patient and their primary provider will 
be notified for further evaluation and management.  
Actigraphy derived 
sleep and physical 
activity data 
Rash on Skin 
Low/Low. Noninvasive measurement of sleep and 
activity data by wearing Actigraph Link on wrist for 
up to 14 days 
Dietary Recall 
Breach of confidentiality 
Low/Low. Study staff will call participants and ask 
about dietary intake for the last 24 hours. The actual 
call will be unannounced but within a specified 2 
week window. Participant will be aware of the 2 week 
window. This will occur 6 times during the study.  
Study Procedure 
Risk 
Likelihood/seriousness/mitigation 
Hyperinsulinemic-
euglycemic clamp 
May cause low blood sugar, 
resulting in hunger, 
sweating, or weakness. 
High/Low. Blood glucose levels will be monitored 
every ten minutes. The participant will be kept 
supine for the visit with bathroom privileges (bedside 
commode/urinal). Glucose will be administered by IV 
at the same time as insulin. After the clamp is 
completed, the participant will be fed a meal and 
kept at the CRU until glucose level meets criteria for 
discharge. 
6,6 2H2 glucose  
infusion 
 
Possible nausea and 
discomfort associated with 
infusion. 
Low/Low. This will be prepared by the Fairview 
Investigational Drug Pharmacy. This is an 
established technique for measuring glucose 

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Protection against Risk 
 We will plan the following to minimize risk: 
 1) Clinical procedures will be performed by properly trained personnel who are 
qualified by training and licensure to perform the procedures. 
 2) Investigator has completed required training re: human subject protections.  
All personnel will comply with all related regulations and laws. 
 3) Subjects will be rigorously screened against inclusion/exclusion criteria to 
ensure that their participation is safe. 
 4) AEs and SAEs will be assessed and followed throughout study; Subjects will 
have contact information to enable them to contact study personnel easily and 
quickly. 
 5) Study data and information will be kept confidential and managed in 
accordance with requirements of HIPAA.  All data will be stored in locked offices 
and not released without subject permission.   
 6) Subjects may discontinue participation at any time, for any reason.   Any 
subject observed to have unacceptable responses to research procedures, or to 
be unable to safely tolerate participation in the study will be withdrawn. 
 7) A data safety monitoring plan will be in place (see appropriate section). 
 8) IRB approval and if needed, IND approval will be obtained (to use stable 
isotopes in healthy humans).  
 9) Monitoring will be conducted by the research team, IRB and independently 
(i.e., at a minimum of annually) by qualified staff of the University of Minnesota’s 
disposal. Subjects will be monitored during 
administration. 
D5Glycerol 
Possible nausea and 
discomfort associated with 
infusion. 
Low/Low. This will be prepared by the Fairview 
Investigational Drug Pharmacy. This is an 
established technique for measuring glucose 
disposal. Subjects will be monitored during 
administration. 
Documentation of 
eating events using 
mCC 
Breach of confidentiality 
Low/Low. All documented eating events (time, 
image) will be stored and sent deidentified as per 
Salk Institute protocols. 
Indirect calorimetry 
Possible discomfort 
associated with canopy 
hood 
Low/Low. A special facemask will be used to 
noninvasively collect breath samples for CO2 and 
O2 to calculate fat and glucose oxidation (RQ). This 
will be for ½ hour for each measurement and the 
participant will be supervised by study staff and CRU 
nursing during this time. 

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Clinical and Translational Science Institute (CTSI) in accordance with the 
established monitoring plan.   
 10) Dr. Chow will allocate adequate time for such monitoring activities.  Dr. 
Chow will also ensure that the monitor or other compliance or quality assurance 
reviewer is given access to all study-related documents and study related facilities 
(e.g. pharmacy, diagnostic laboratory, etc.), and has adequate space to conduct 
the monitoring visit. 
13.2 Reproduction Risks: N/A 
13.3 Risks to Others: N/A 
14.0 Potential Benefits to Participants 
14.1 Potential Benefits: There are no guarantee of benefits. However, there is 
potential benefit to the human subjects would be weight-loss with the dietary 
intervention. Since we are deliberately enrolling obese, insulin resistant humans, 
their physical health may benefit from any observed weight loss. The subjects are 
allowed to withdraw from the study at any time per their request or per the 
judgment of the investigator. 
15.0 Statistical Considerations 
15.1 Data Analysis Plan:  
Statistical Analysis Plan 
Participants’ demographic and characteristics at baseline will be summarized and 
compared between groups using Chi-square test or Fisher’s exact test for 
categorical variables and analysis of variance (ANOVA) F test for continuous 
variables. Baseline characteristics that are significantly different between groups 
will be considered as adjusters in primary analyses. Pre-post changes in weight 
and body composition (Aim 1); caloric intake, diet quality, physical activity, sleep 
quality and duration, and fat oxidation  (Aim 2); and metabolic flexibility, insulin 
sensitivity, and lipolysis (Aim 3) will be compared between groups using ANOVA F 
test.  If the overall F test is significant, pairwise comparisons will be conducted 
with Tukey’s method to adjust for multiple comparisons. In addition, because 
weight and diet quality are measured at three time points we will also investigate 
temporal trends using linear mixed effects models with subject-specific random 
intercepts. Data distributions will be checked before conducting statistical tests. 
Transformation will be done if the normality assumption is not met. We will 
investigate how sex and other clinical factors, such as age, influence change in 
weight and body composition, caloric balance, and metabolic flexibility by 
including relevant clinical factors into our models. Secondary analysis will be 
conducted to examine the association between achieved fasting duration and 
pre-post change in outcomes, using multivariate linear regression models. Models 
will include effects of achieved fasting duration, treatment group, and the 

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interaction between both effects. Analyses will be performed in R or SAS (Version 
9.4, SAS Institute Inc., Cary, NC). We will also analyze by intention to treat and per 
protocol. 
Missing data management: 
If the participants want to discontinue their dietary intervention, we will offer the 
following options in a sequential fashion: 1) Address any limiting factors that may 
prevent program completion, 2) Move the final assessment sooner to be 
performed at the time of dropping out, 3) Continue data collection as intensively 
as possible given the participant’s preferences. We will also collect reasons for 
drop out (i.e. time/duration, transportation, side effects, perceptions of efficacy, 
interaction with staff) to use as potential covariates in the statistical model.  In 
terms of missing data, results from the last observation carried forward as well as 
multiple imputation analysis32 For the final analysis, intention to treat analysis 
and per-protocol analysis (analysis in those who completed the protocol) will be 
performed. 
15.2 Power Analysis:  
Sample size determination is based on the primary outcome in Aim 1: weight loss 
from baseline to 12 weeks. Based on the preliminary data and the literature,100 
we anticipate weight loss of 3.6 kg (SD of 1.9) in the TRE group, 1.5 kg (SD of 2.4) 
in the non-TRE group, and 4 kg (SD of 2.5) in the CR group. With n=24 per group 
and alpha = 0.05/3 to adjust for multiple comparisons, we will have 80% power to 
detect the described difference in weight loss between the TRE group vs. the non-
TRE group, 85% power to detect the described difference in weight loss between 
the CR group vs. the non-TRE group, and 83% power to detect a difference of 1 SD 
between groups in pre-post change in other outcomes including body 
composition (Aim 1), caloric intake (Aim 2), and metabolic flexibility (Aim 3). 
Based on the preliminary data, the SD is 1.5, 1.4, and 0.3 kg for change in fat 
mass, lean mass, and visceral fat, respectively. The SD for change in energy intake 
is estimated to be 254 kcal/d and the SD for metabolic flexibility is estimated to 
be 0.05.7, 101 Conservatively accounting for a 20% dropout rate, we will enroll 90 
subjects (30/group). 
15.3 Statistical Analysis:  
Participants’ demographic and characteristics at baseline will be summarized and 
compared between groups using Chi-square test or Fisher’s exact test for 
categorical variables and analysis of variance (ANOVA) F test for continuous 
variables. Baseline characteristics that are significantly different between groups 
will be considered as adjusters in primary analyses. Pre-post changes in weight 
and body composition (Aim 1); caloric intake, diet quality, physical activity, sleep 
quality and duration, and fat oxidation  (Aim 2); and metabolic flexibility, insulin 
sensitivity, and lipolysis (Aim 3) will be compared between groups using ANOVA F 

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test.  If the overall F test is significant, pairwise comparisons will be conducted 
with Tukey’s method to adjust for multiple comparisons. In addition, because 
weight and diet quality are measured at three time points we will also investigate 
temporal trends using linear mixed effects models with subject-specific random 
intercepts. Data distributions will be checked before conducting statistical tests. 
Transformation will be done if the normality assumption is not met. We will 
investigate how sex and other clinical factors, such as age, influence change in 
weight and body composition, caloric balance, and metabolic flexibility by 
including relevant clinical factors into our models. Secondary analysis will be 
conducted to examine the association between achieved fasting duration and 
pre-post change in outcomes, using multivariate linear regression models. Models 
will include effects of achieved fasting duration, treatment group, and the 
interaction between both effects. Analyses will be performed in R or SAS (Version 
9.4, SAS Institute Inc., Cary, NC)  
15.4 Data Integrity:  
1. Rigor: Recruitment of participants will be performed using the defined 
inclusion/exclusion criteria, with particular efforts devoted towards even 
distributions of men/women and inclusion of minority populations. Data collected 
at the initial and final study visits will be entered by research staff into Research 
Electronic Data Capture (REDCap), which uses a MySQL database via a secure web 
interface with data checks used during data entry to ensure data quality.  Data 
summaries and quality control checks will be run routinely.  
2. Transparency: All data obtained in this project will be de-identified with each 
study subject receiving a unique study ID number. The de-identified data will be 
routinely shared among team members and discussed at regular meetings (Table 
1) to evaluate recruitment status and the next step of experiments, analyses, and 
interpretation of the findings. Results of our studies will be submitted for 
publication in peer reviewed journals, and NIH requirements for access of 
manuscripts through PubMed Central will be fulfilled. 
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 and 
therefore assert HIPAA does not apply.   
 
☒ 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). 

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☐ 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:  
☐ An external IRB (e.g. Advarra) is reviewing and we are requesting use of the 
authorization language embedded in the template consent form in lieu of the 
U of M stand-alone HIPAA Authorization.  Note: External IRB must be serving 
as the privacy board for this option. 
 
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:  
 
16.3 
Only patients who have authorized their participation in research in EPIC and 
have not opted out of research will be provided by BPIC for us to review for our 
study recruitment. 
16.4 
Approximate number of records required for review: 
Up to 2.8 million (EPIC EHR records). This will be done by BPIC.  Only patients who have 
authorized their participation in research in EPIC will be reviewed. We anticipate that ~ 
2000 records will be referred to us for potential contact for study recruitment. 
 
16.5 
Please describe how you will communicate with research participants during 
the course of this research.  Check all applicable boxes 

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☐ 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. 
 
  This communication will include the following: 1) phone calls, 2) letters, 3) 
texting and 4) Email. We will communicate with participants via telephone 
(using a study-specific phone number) for pre-screening, appointment 
scheduling, appointment reminders, etc. We will also communicate through 
text message or unsecure email if the participant agrees in writing by signing 
the GUIDELINES AND CONSENT FOR TEXT MESSAGE CORRESPONDENCE FOR 
RESEARCH PARTICIPANTS and/or GUIDELINES AND CONSENT FOR 
UNSECURED EMAIL CORRESPONDENCE FOR RESEARCH PARTICIPANTS. 
16.6 
Explain how the research team has legitimate access to patients/potential 
participants: The research team will be permitted to access sources of private 
information because all participants will be required to sign a HIPAA waiver at the time 
of informed consent. 
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) 

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 ☒ Store 
☒ Analyze 
☒ Share 
☒ In an AHC-IS supported server. Provide folder path, location of server and IT 
Support Contact: 
MedDerm(\\med.ahc.umn.edu\med)(N:)\Chow Group 
 ☒ Store 
☒ Analyze 
☒ Share 
☒ In an AHC-IS supported desktop or laptop.  
Provide UMN device numbers of all devices: 20181216 
 ☒ Store 
☒ Analyze 
☒ Share 
☒ Other. Describe:  
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 
☐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: 
AHC tablet to input survey data (20181716) 
16.8 Consultants. Vendors. Third Parties.  
Electronic data will be shared with our external collaborators using the UMN Box 
account. We will also use the UMN Box account to store results from our external 
collaborators.  All samples sent to the external collaborators (i.e. electronic 
information to Dr. Panda, blood samples to Dr. Bergman) will be sent de-
identified. These samples will be analyzed and stored in their laboratories as per 
their institutional protocol. Results from our external collaborators will be placed 
into the UMN Box account (as per above) for our analysis. 
16.9 Links to identifiable data: There is be a participant unique study ID established in 
REDCap, which will link participants to the data. This will be used to identify the 
data with the REDCap data.  All shared data will be deidentified. 
 
16.10 Sharing of Data with Research Team Members. Data between study members at 
the University of Minnesota will be shared by phone, voice mail, U of MN email, 
BOX, centralized shared drive, and REDCap. 
 

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16.11 Storage and Disposal of Paper Documents: In accordance with NIH policy, all 
study documents will be maintained for at least 3 years ater the study ends and 
for a longer time if required by University of Minnesota policy. All signed and 
dated HIPAA authorizations and consent documents that include HIPAA 
authorizations wil be maintained for at least 6 years after completion of the 
study. After this time period, all research records will be destroyed. Computer 
files will be deleted and hard copy materials will be discarded in accordance 
with University of Minnesota policy. 
17.0 Confidentiality 
17.1 Data Security:  
• Training: All study staff will be appropriately trained in data security. 
• Authorization of access: Only designated IRB-approved staff will have 
access to the data. 
• Password protection/encryption/physical controls: All data will be stored 
in REDCap. 
• Certificates of confidentiality:  Since this study will be funded by an NIH 
RO1 Grant, we will apply for a NIH Certificate of Confidentiality. All entities 
that are part of this study will be subject to the requirements of this 
Certificate. 
• Separation of Identifiers: Study staff will keep the mapping of 
identification code to the identity of the participant in a database 
protected by two-levels of password protection stored separately from 
the data on Box. 
• A Certificate of Confidentiality is automatically issued by the NIH For this 
project. 
18.0 Provisions to Monitor the Data to Ensure the Safety of Participants 
 
18.1 Data Integrity Monitoring. Monitoring will be conducted by study staff, IRB and 
independently (i.e. at least annually, as a minimum) by qualified staff of the 
University of Minnesota’s Clinical and Translational Science Institute (CTSI) in 
accordance with the established monitoring plan.  Monitoring events will include 
the following: Study recruitment, subject compliance with visits, subject accrual, 
adverse events, stopping rules with regard to enrollment/drop out/missing 
data/adverse events.  
The data for each subject will be entered into a REDCap database. The REDCap 
database uses a MySQL database via a secure web interface with data checks 
used during data entry to ensure data quality. REDCap includes a complete suite 
of features to support HIPAA compliance, including a full audit trail, user-based 
privileges, and integration with the institutional LDAP server. The MySQL 

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database and the web server will both be housed on secure servers operated by 
the University of Minnesota Academic Health Center’s Information Systems group 
(AHC-IS). The servers are in a physically secure location on campus and are 
backed up nightly, with the backups stored in accordance with the AHC-IS 
retention schedule of daily, weekly, and monthly tapes retained for 1 month, 3 
months, and 6 months, respectively. Weekly backup tapes are stored offsite. The 
AHC-IS servers provide a stable, secure, well-maintained, and high-capacity data 
storage environment, and both REDCap and MySQL are widely-used, powerful, 
reliable, well-supported systems. Access to the study's data in REDCap will be 
restricted to the members of the study team by username and password. 
The clinical data will be reviewed weekly by the study team to ensure progress, 
timely data entry and addressing missing data. The clinical data will also be 
reviewed monthly by Dr. Chow to double-check proper data entry and missing 
data. 
Frequency of Monitoring 
Data Type 
Frequency of Review 
Study recruitment 
Monthly 
Subject compliance with visits 
As event occurs and 
weekly 
Subject accrual (adherence to protocol regarding demographics, 
inclusion/exclusion) 
Quarterly 
Achievement of study milestones 
Quarterly 
Adverse event rates 
As event occurs and 
quarterly 
Stopping rules with regards to enrollment/drop out/missing data 
Yearly 
Patient safety officer 
As event occurs and 
quarterly 
 
18.2 
Data Safety Monitoring 
Recording of Adverse Event:  
 
At each contact with the subject, the investigator and study staff must seek 
information on adverse events by specific questioning and, as appropriate, by examination. 
Information on all adverse events should be recorded immediately in the source document, 
and also in the appropriate adverse event module of the case report form (CRF).  All clearly 
related signs, symptoms, and abnormal diagnostic procedures results should be recorded in 
the source document, though should be grouped under one diagnosis. 
 
All adverse events occurring during the study period must be recorded. The clinical 
course of each event should be followed until resolution, stabilization, or until it has been 
determined that the study participation is not the cause.  Serious adverse events that are still 

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ongoing at the end of the study period must be followed up to determine the final outcome.  
Any serious adverse event that occurs after the study period and is considered to be possibly 
related to the study participation should be recorded and reported immediately. 
Reporting of Serious Adverse Events 
 
A serious adverse event is any adverse event that is: 1) Fatal 2) Life Threatening 3) 
Requires or prolongs a hospital stay 4) Results in persistent or significant disability or 
incapacity or 5) A congenital anomaly or birth defect. Of note, important medical events are 
events that may not be immediately life-threatening, but are clearly of major clinical 
significance and may be a serious adverse event. They may jeopardize the subject, and may 
require intervention to prevent one or the other serious outcomes noted above. 
Adverse Event Reporting Plan:  
 
Reporting of adverse events will follow University of Minnesota IRB requirements. 
Within 48 hrs of any serious adverse event, a verbal or email report will be made to IRB 
followed by a detailed written report within 10 business days. 
Patient safety officer: 
 
We will have Dr. Amir Moheet serve as the patient safety officer for the study. He will 
review any adverse events related to the study, independent of the IRB and the clinical trials 
monitor. His report within be submitted to the IRB. His focus will be to provide independent 
medical opinion on study associated adverse events. 
IRB Notification by Investigators 
 
Reports of all serious adverse events (including follow-up information) must be 
submitted to the IRB within 10 working days if it falls under the UPIRTSO guidelines.  Copies of 
each report and documentation of IRB notification and receipt will be kept in the Clinical 
Investigator’s binder 
Unanticipated Problems Involving Risk To Subjects or Others (UPIRTSO) Events 
Investigators are required to submit a report of UPIRTSO events to the IRB within 10 
working days of first learning of the event. 
Responsible Individual(s) or Group 
 
Monitoring will be conducted by the PI, IRB and independently (i.e. at least annually as 
a minimum) by qualified staff of the University of Minnesota’s Clinical and Translational 
Science Institute (CTSI, supported by the CTSA grant awarded in 2018) in accordance with the 
established monitoring plan. 
Frequency of Monitoring 
Data Type 
Frequency of Review 
Study recruitment 
Monthly 
Subject compliance with visits 
As event occurs and 
weekly 

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Subject accrual (adherence to protocol regarding demographics, 
inclusion/exclusion) 
Quarterly 
Achievement of study milestones 
Quarterly 
Adverse event rates 
As event occurs and 
quarterly 
Stopping rules with regards to enrollment/drop out/missing data 
Yearly 
Patient safety officer 
As event occurs and 
quarterly 
 
19.0 Provisions to Protect the Privacy Interests of Participants 
19.1 Protecting Privacy:  
The study consent form will describe in detail any intrusive, uncomfortable, or 
unfamiliar questions, procedures, or interactions with researchers or study 
personnel that the participant will be asked to complete. Furthermore, the study 
consent form will communicate that it is the participant’s right to opt-out of any 
study procedures or the study as a whole or withdraw from the study at any time 
and this information will be reiterated and revisited periodically throughout the 
study in advance of intrusive, uncomfortable, or unfamiliar questions procedures 
or interactions. Participants will not be compelled or pressured to provide 
information or specimens or study data that they do not wish to provide. 
19.2 Access to Participants:  
Participants have been fully informed of the ways in which their data will/may be 
used during the informed consent process. The research team has been trained in 
conducting these conversations and the participants are also assessed for their 
understanding of consent prior to signing the consent form or initiating any study 
procedures. 
20.0 Compensation for Research-Related Injury 
20.1 Compensation for Research-Related Injury:  
In the event that research-related activities result in an injury, treatment will be 
provided to the participant (e.g., first aid, emergency treatment, and follow-up 
care as needed). Care for such injuries will be billed in the ordinary manner to the 
participant or the participant’s insurance company 
20.2 Contract Language: N/A 
21.0 Consent Process 
21.1 Consent Process (when consent will be obtained):  
Consent forms describing in detail the study, study procedures, and risks are 
given to the participant and documentation of informed consent is required prior 

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to any research procedures via written signatures or esignatures using REDCap. 
The following consent materials are submitted with this protocol: 
Consent will take place via a virtual visit using Zoom or Doximity. Participants will 
have the opportunity to carefully review the written consent form and ask 
questions prior to signing. The participants will be given the opportunity to review 
the consent with family and caregivers and return on a subsequent day to sign 
the consent and enroll in the study. 
  
Informed consent is a process that is initiated prior to the individual’s agreeing to 
participate in the study and continues throughout the individual’s study 
participation. Extensive discussion of risks and possible benefits of participation 
will be provided to the participants and their families. Consent forms will be IRB-
approved and the participant will be asked to read and review the document. The 
investigator will explain the research study to the participant and answer any 
questions that may arise. We will use the teach-back method to assess the 
participant’s understanding of the study.  
  
All participants will receive a verbal explanation in terms suited to their 
comprehension of the purposes, procedures, and potential risks of the study and 
of their rights as research participants. Participants will have the opportunity to 
carefully review the written consent form and ask questions prior to signing. The 
participants should have the opportunity to discuss the study with others or think 
about it prior to agreeing to participate. The participant will sign the informed 
consent document prior to any procedures being done specifically for the study. 
The participants may withdraw consent at any time throughout the course of the 
trial. A copy of the informed consent document will be given to the participants 
for their records. The rights and welfare of the participants will be protected by 
emphasizing to them that the quality of their medical care will not be adversely 
affected if they decline to participate in this study. 
 
21.2 Waiver or Alteration of Consent Process (when consent will not be obtained): We 
will obtain verbal consent over the phone for subject to be fasting for the first 
visit. 
21.3 Waiver of Written/Signed Documentation of Consent (when written/signed 
consent will not be obtained):  
We will ask the IRB to approve a Waiver of Signed Documentation of Consent for 
the phone screening to determine eligibility and to explain to the potential 
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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 
22.0 Setting 
22.1 Research Sites:  
Fairview Health System Electronic Health Record (EHR):  
The University of Minnesota is a partner of the Fairview Health System which 
uses a modern enterprise level EHR (EPIC) since 2011. As of January 2019 the 
Fairview EHR has ~2.8 million living patients. Of these patients, 1,700 had 
prediabetes diagnosed either as fasting glucose between 100-125 (inclusive) or 
HbA1c between 5.7-6.4 (inclusive)  within the last 3 months (since February 2019) 
and have given consent for participation. 
 
UMMC Central Lab 
Blood work, other than saved serum/ plasma samples, will be processed at the 
UMMC main lab and results faxed to the PI.  
Clinical and Translational Science Institute (CTSI), University of Minnesota 
The Clinical and Translational Science Institute (CTSI) at the University of 
Minnesota offers comprehensive research support for clinical investigators, from 
concept through publication. CTSI supports all stages of the research process, 
providing research and regulatory support, specialized facilities, and analytical 
services. Concept and pre-study services include assistance with study design 
(biostatistical support), protocol and budget development, study population 
definition (i2b2/SHRINE resources), bioinformatics support, investigational new 
drug (IND) or investigational device exemption (IDE) application assistance, 
contract negotiations, institutional committee’s approval assistance (IRB, 
Radiation Safety, Institutional Biosafety, etc.), and clinical trial registration with 
ClinicalTrials.gov. CTSI also offers educational and training opportunities for new 
investigators and study coordinators. Strong relationships across the University’s 
Academic Health Center provide access to extensive expertise and collaborative 
opportunities. Biological specimen handling, storage and retrieval facilities and 
software (caTissue) are available within the University.  
During the clinical study, both inpatient and outpatient adult clinical spaces are 
available in several facilities. Dr. Chow will be doing most of her clinical research 

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work at the Clinical Research Unit (CRU), a 16,250 square foot inpatient facility 
including 10 inpatient beds, 4 outpatient beds, rooms dedicated to glucose clamp 
studies, rooms for human performance studies with specialized equipment for 
exercise testing, non-invasive vascular investigation and body composition 
assessments. The CRU is directly connected to the University of Minnesota 
Medical Center, Fairview and the office building (Phillips Wangensteen Building) 
where Dr. Chow has her office. The Delaware Clinical Research Unit (DCRU), a 
36,750 square foot outpatient facility including separate adult and pediatric 
oriented facilities: Adult facilities include: 10 exam rooms, 4 consultation rooms, 5 
specialized rooms, a metabolic kitchen, sample acquisition room and on-site 
laboratory. The facility provides 23 free parking spaces for research participants. 
The DCRU is within 2 blocks of the CRU. 
o 
Implementation staffing (research project managers, research nurses 
and certified medical assistants, clinical research coordinators, clinical 
trial monitors, a registered dietitian, laboratory technician, and other 
specialized technical services) can be provided to the extent needed 
by the investigator. A state of the art iDXA for bone density and body 
composition is available on site. Trial monitoring, and ongoing 
regulatory reporting and support are provided. The CTSI provides 
comprehensive clinical research facilities and support across specialty 
areas, tailored to the needs of the investigator, to facilitate effective 
and efficient use of resources without unnecessary duplication. 
LIHP Facilities 
The LIHP, directed by Dr. Dengel, occupies 4500 square feet in Room 141 of 
Mariucci Arena on the University of Minnesota campus. A University of 
Minnesota parking lot is located adjacent to the LIHP and provides easy parking 
for participants. The LIHP houses the Hologic Horizon A iDXA, where the body 
composition will be measured. There is a separate male and female restroom 
located next to the LIHP.  
Nutrition Coordinating Center:   
The Nutrition Coordinating Center (NCC) is located in the Division of Epidemiology 
and Community Health, and occupies about 6,000 square feet of office space. Dr. 
Lisa Harnack (Co-I) is the Director of the NCC since 2007. The NCC includes office 
space for staff, telephone calling stations, resource area, training room, and 
conference rooms. The NCC has developed and maintains a research-quality food 
and nutrient database linked to computerized interactive interview software, the 
Nutrition Data System for Research (NDSR). The NDSR is designed primarily for 
clinical research and epidemiological studies investigating relationships between 
diet and health.  The NDSR dietary data collection and nutrient analysis tool is 
well recognized in the nutrition research community and has been licensed for 

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use in hundreds of research studies. Clients may also choose to enlist the services 
of NCC to accomplish some or all of their dietary data collection and processing 
objectives. The NCC Service Center offers training and certification of dietary 
interviewers, collection of dietary intake by telephone interview, processing of 
food records, analysis of menu and recipe data, development of dietary data 
collection protocols, and customized support services for additional related 
research needs. The Service Center has provided the aforementioned types of 
services to hundreds of studies since 1974, including carrying out entry and 
nutrient analysis of food records obtained via meal observation procedures 
similar to those to be utilized in the proposed study. 
 
 
Center for Magnetic Resonance Research 
Dr. Ryder will also utilize the Center for Magnetic Resonance Research (CMRR for 
measurement of liver fat content via MRI collected during the study. The CMRR is 
fully furnished and equipped for modern translational and clinical research. The 
facility house multiple ultrahigh magnetic fields (7 Tesla and above) but also 
contains three 3 Tesla (Siemens, Berlin, Germany) scanners which will be used in 
this study. The 3 Tesla Trio (Siemens) system is a standard system similar to those 
used in hospitals for clinical diagnosis. This system can be used for standard 
imaging or spectroscopy measurements of liver fat. The CMRR is equipped with 
extensive computation resources, including onsite file servers and compute 
servers are located in a modern secured server room with diesel-backed UPS 
power and fully redundant cooling. The center also includes subject rooms for 
consenting participants, or other participant needs. The center provides MR 
Technologists to run the 3T scanners, assist with data collection, and maintain 
equipment and troubleshoot scanner issues. 
 
Salk Institute: 
Environment. The Salk Institute offers an excellent intellectual environment for 
the scientific success of its faculty. Dr. Panda’s lab is part of the Regulatory 
Biology Laboratories and is housed in the same floor as the Laboratory of 
Genetics. The open lab structure of the institute offers constant scientific 
exchange among researchers and PIs. Open lab design of the Salk Institute fosters 
persistent interactions among these lab members. Panda lab has ongoing 
collaborations with several Salk Institute faculty in the field of metabolism, 
endocrinology, and data science. They include Ron Evans, Marc Montminy, 
Reuben Shaw, Saket Navlakha, Alan Saghatelian with whom Panda lab has several 
active collaboration projects. These colleagues and their scientific staffs offer a 
productive intellectual environment relevant to the success of this project. 

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Laboratory. Laboratory space for 10 full time researchers furnished with standard 
laboratory equipment (thermocyclers, benchtop centrifuges, dissecting 
microscopes, DNA/RNA, protein gel electrophoresis apparatus, heat blocks, water 
baths, freezers, refrigerators, tissue culture hoods, tissue culture incubators, Q-
PCR, FPLC) is available within the department of Regulatory biology at Salk 
institute. 
 
Clinical. The lab has developed, validated, and deployed the myCircadianClock 
app for monitoring and intervening human eating pattern. The app is hosted on 
secured HIPAA compliant Amazon Web Server (AWS). The database and the 
backend processes communicate with the user’s smartphone using double 
encryption. 
 
Computer. The laboratory has 12 recent model PCs, and 3 Macintosh computers. 
In addition, shared facilities at Salk include high-end personal computers for 
image processing, database searches, and modeling. Salk institute’s central 
Research Computing department maintains an extensive data communications 
network available to all laboratories. 
 
Office. The PI has a 100 sq. ft. office located adjacent to the laboratory; desk with 
computer are available for staff associated with the project. 
 
Data analysis only facilities – This will be using deidentified data 
Dr. Mashek’s laboratory 
Dr. Mashek’s lab is approximately 1600 ft2 plus two adjacent 125 ft2 rooms for 
tissue culture and radioisotope studies. The laboratory is equipped with a tissue 
culture hood, two fume hoods, an autoclave and a walk-in cold room. The 
following equipment are either in Dr. Mashek’s laboratory or is readily accessible: 
Class 2, Type A2 biological safety cabinet, NuAire, Inc.; water jacketed CO2 
incubator, NuAire, Inc.; Nucleofector II, Amaxa Biosystems, Inc.; Synergy HT multi-
detection microplate reader, BIO-TEK Instruments, Inc.; FLUOstar OPTIMA 
microplate reader, BMG Labtech; Mini-Protean 3 Cell and Trans-Blot 
electrophoretic transfer Cell, BIO-RAD; PowerPac HC Power Supply, BIO-RAD; 
CKX41SF2 inverted microscope, Olympus; IX70 inverted microscope with 
fluorescence and imaging capabilities, Olympus; Labphoto 2 microscope, Nikon; 
Saturn 5 GC/MS, Varian; PTC-200 DNA Engine Cycler, BIO-RAD; ABI StepOne Plus 
Real-Time PCR system, Applied Biosystems; SMARTSPEC Plus, BIO-RAD; Spinchron 

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R refrigerated centrifuge, Beckman Instruments; RT6000B refrigerated centrifuge, 
Sorvall; J2-21 centrifuge, Beckman; Orion Star Series pH meter, Thermo Electron; 
L8-70 Ultracentrifuge, Beckman; Ultralow temperature freezer, Sanyo; AQT series 
top loading balance, Adams Equipment; A30 analytical balance, Mettler, Inc.; G24 
environmental incubator shaker, New Brunswick Scientific Co., Inc; Gyrotory 
water bath shaker-model G76, New Brunswick Scientific Co., Inc.; general purpose 
water bath, Precision, Seahorse XF-96, Seahorse Bioscience. 
Coordinating Centers for Biometric Research (CCBR) and Biostatistical Design & 
Analysis Center (BDAC) 
Several SPARC servers (Solaris operating system) and Intel servers (Linux and 
FreeBSD operating systems) provide application service to the CCBR and the 
Division of Biostatistics. Access to these applications is provided by more than 
100 thin-client (X-servers) desktops. All servers are connected to the Internet. The 
CCBR machines have more than 20 terabytes of disk storage available. Statistical 
analysis and data management are the primary applications provided by the 
servers. Statistical analyses are performed using SAS, SAS/GRAPH, SAS/IML, and R. 
Oracle is the primary relational database management system. Various SQL clients 
are used including SAS, Perl, SQL*Plus, and PHP.
Bryan Bergman’s laboratory 
The facilities and other resources available to the research team include 
everything needed to complete the proposed studies. Dr. Bergman has been 
performing human research studies similar to the one proposed for 18 years at 
this institution, so the infrastructure and collaborations are ready to successfully 
complete this project. In addition to Dr. Bergman’s laboratory, there is an NIH 
funded Clinical Translational Research Center (CTRC) at the University of Colorado 
Anschutz Medical campus (UCAMC) for all human work proposed. The intellectual 
environment is filled with other NIH funded investigators who are performing 
work complimentary to what is proposed, and there is a strong history of human 
clinical research and funding at our institution. Together, these facilities available 
to the research team provide a scientific environment that strongly supports the 
proposed research and, therefore, collectively contributes to the success of the 
project. 
Dr. Bergman has approximately 1000 sq. ft. of wet laboratory space located in 
the School of Medicine to support the proposed research. He has all the 
necessary equipment and expertise to perform muscle dissection, western 
analyses, cell culture experiments, and tissue incubation required for these 
analyses as they are standard procedures in the laboratory. Specifically, he has a 
fume hood, an Organomation 100 position multivap nitrogen evaporator, 
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refrigerators, Mettler-Toledo analytical balance, pH meter, gel rocker, BioRad 
power supplies and western equipment. Additionally, Dr. Bergman has half of a 
120 sq. ft cold room, and a 200sq. ft. cell culture laboratory across the hall from 
his laboratory containing two incubators, an inverted microscope, water bath, 
refrigerator, centrifuge, and a cell culture hood.  Dr. Bergman’s GC/MS and LC/MS 
instruments are on the 6th floor of the building containing his laboratory, in a 
space designated as core space for the Colorado Nutrition Obesity Research 
Center. Importantly, he has all the necessary internal standards and bone fide 
standards required to identify and quantify neutral lipids including diacylglycerol 
isomers, polar lipids such as acyl-carnitines, as well as deoxysphingolipids via 
LC/MS. These analyses are routine in the Bergman laboratory. o 
Local 
scientific and ethical review structure. 
22.2 International Research: N/A 
23.0 Multi-Site Research 
N/A 
24.0 Coordinating Center Research 
N/A 
25.0 Resources Available 
25.1 
Resources Available: 
Division of Endocrinology, Department of Medicine, University of Minnesota (Primary 
location Dr. Lisa Chow, PI, Contact PI) 
Office: 
The Division of Endocrinology has adequate office space for this project. Each faculty 
member has an individual office and computer assigned for their use. Modern office 
equipment such as facsimile machines, photocopiers, postage machine, and mailroom 
services will be available to this project.  
Computer Services: 
The Division of Endocrinology is part of the Academic Health Center (AHC) and has AHC 
support for computer maintenance and centralized backup of computer files.  
Clinical and Translational Science Institute (CTSI), University of Minnesota 
The Clinical and Translational Science Institute (CTSI) at the University of Minnesota 
offers comprehensive research support for clinical investigators, from concept through 
publication. CTSI supports all stages of the research process, providing research and 
regulatory support, specialized facilities, and analytical services. Concept and pre-study 
services include assistance with study design (biostatistical support), protocol and 
budget development, study population definition (i2b2/SHRINE resources), 

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bioinformatics support, investigational new drug (IND) or investigational device 
exemption (IDE) application assistance, contract negotiations, institutional committee’s 
approval assistance (IRB, Radiation Safety, Institutional Biosafety, etc.), and clinical trial 
registration with ClinicalTrials.gov. CTSI also offers educational and training 
opportunities for new investigators and study coordinators. Strong relationships across 
the University’s Academic Health Center provide access to extensive expertise and 
collaborative opportunities. Biological specimen handling, storage and retrieval 
facilities and software (caTissue) are available within the University.  
During the clinical study, both inpatient and outpatient adult clinical spaces are 
available in several facilities. Dr. Chow will be doing most of her clinical research work 
at the Clinical Research Unit (CRU), a 16,250 square foot inpatient facility including 10 
inpatient beds, 4 outpatient beds, rooms dedicated to glucose clamp studies, rooms for 
human performance studies with specialized equipment for exercise testing, non-
invasive vascular investigation and body composition assessments. The CRU is directly 
connected to the University of Minnesota Medical Center, Fairview and the office 
building (Phillips Wangensteen Building) where Dr. Chow has her office. The Delaware 
Clinical Research Unit (DCRU), a 36,750 square foot outpatient facility including 
separate adult and pediatric oriented facilities: Adult facilities include: 10 exam rooms, 
4 consultation rooms, 5 specialized rooms, a metabolic kitchen, sample acquisition 
room and on-site laboratory. The facility provides 23 free parking spaces for research 
participants. The DCRU is within 2 blocks of the CRU. 
Implementation staffing (research project managers, research nurses and certified 
medical assistants, clinical research coordinators, clinical trial monitors, a registered 
dietitian, laboratory technician, and other specialized technical services) can be 
provided to the extent needed by the investigator. A state of the art iDXA for bone 
density and body composition is available on site. Trial monitoring, and ongoing 
regulatory reporting and support are provided. The CTSI provides comprehensive 
clinical research facilities and support across specialty areas, tailored to the needs of 
the investigator, to facilitate effective and efficient use of resources without 
unnecessary duplication. 
Data for this study will be entered into a REDCap database. The REDCap database uses 
a MySQL database via a secure web interface with data checks used during data entry 
to ensure data quality. REDCap includes a complete suite of features to support HIPAA 
compliance, including a full audit trail, user-based privileges, and integration with the 
institutional LDAP server. The MySQL database and the web server will both be housed 
on secure servers operated by the University of Minnesota Academic Health Center’s 
Information Systems group (AHC-IS). The servers are in a physically secure location on 
campus and are backed up nightly, with the backups stored in accordance with the 
AHC-IS retention schedule of daily, weekly, and monthly tapes retained for 1 month, 3 
months, and 6 months, respectively. Weekly backup tapes are stored offsite. The AHC-
IS servers provide a stable, secure, well- maintained, and high-capacity data storage 

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environment, and both REDCap and MySQL are widely-used, powerful, reliable, well-
supported systems. Access to the study's data in REDCap will be restricted to the 
members of the study team by username and password. 
Fairview Health System Electronic Health Record (EHR): 
The University of Minnesota is a partner of the Fairview Health System which uses a 
modern enterprise level EHR (EPIC) since 2011. As of January 2019 the Fairview EHR 
has ~2.8 million living patients. Of these patients, 1,700 had prediabetes diagnosed 
either as fasting glucose between 100-125 (inclusive) or HbA1c between 5.7-6.4 
(inclusive)  within the last 3 months (since February 2019) and have given consent for 
participation. Data analysis of the EHR will be through BPIC.  
Department of Kinesiology 
Dr. Don Dengel (Co-I) 
Office:  
The School of Kinesiology has adequate office space for this project. Each faculty 
member has an individual office and computer assigned for their use. Modern office 
equipment such as facsimile machines, photocopiers, postage machine, and mailroom 
services will be available to this project. The School of Kinesiology is part of the College 
of Education and Human Development (CEHD) and has CEHD support for computer 
maintenance and centralized, secure backup of computer files. 
Relevant resources available to Dr. Dengel:  Laboratory of Integrative Human 
Physiology (LIHP): 
LIHP staff 
Currently, Dr. Dengel directs the LIHP, which provides vascular, exercise and body 
composition testing.  His research group consists of one laboratory coordinator and 6 
graduate students. If this proposal is funded, he will use current facilities, equipment 
and infrastructure to facilitate implementation of the indirect calorimetry and DXA 
analysis. Dr. Dengel’s laboratory coordinator will perform the DXAs and indirect 
calorimetry measurements for this study.   
LIHP Facilities 
The LIHP, directed by Dr. Dengel, occupies 4500 square feet in Room 141 of Mariucci 
Arena on the University of Minnesota campus. A University of Minnesota parking lot is 
located adjacent to the LIHP and provides easy parking for participants. The LIHP 

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houses the Hologic Horizon A DXA where the body composition will be measured. 
There is a separate male and female restroom located next to the LIHP.  
Assessments performed at LIHP 
Body Composition.  
The LIHP has a Body Composition Testing Laboratory which contains equipment for the 
determination of body composition including bone mass, fat mass and lean body mass.  
At baseline and 12 weeks, all subjects will have their height and weight will be 
determined using a wall-mounted stadiometer and an electronic scale.  Body mass 
index (BMI) will be calculated as the body weight in kg/height2 (m2).  Percent body fat 
and lean muscle mass will be determined by Hologic Horizon A (Hologic Inc., 
Marlborough, MA) dual X-ray absorptiometer (DXA). The DXA will provide bone 
densities and calculations of total body fat, lean body mass and bone mass. The Hologic 
Horizon A DXA can accommodate individuals who weigh up to 500 lbs and will provide 
calculations of total body fat, regional fat, lean body mass, and bone mass. The Hologic 
Horizon A can provide adipose measures of the android and gynoid regions as well as 
estimation of visceral adipose tissue.1 Prior to daily testing the Hologic Horizon A DXA 
is calibrated using a phantom of known composition. The Hologic Horizon A DXA 
undergoes a yearly preventative diagnostic evaluation.  
Database for Indirect Calorimetry and DXA test results 
The indirect calorimetry and DXA data for each subject will be entered into a REDCap 
database by laboratory coordinator immediately after each test. The REDCap database 
uses a MySQL database via a secure web interface with data checks used during data 
entry to ensure data quality. REDCap includes a complete suite of features to support 
HIPAA compliance, including a full audit trail, user-based privileges, and integration 
with the institutional LDAP server. The MySQL database and the web server will both 
be housed on secure servers operated by the University of Minnesota Academic Health 
Center’s Information Systems group (AHC-IS). The servers are in a physically secure 
location on campus and are backed up nightly, with the backups stored in accordance 
with the AHC-IS retention schedule of daily, weekly, and monthly tapes retained for 1 
month, 8 weeks, and 16 weeks, respectively. Weekly backup tapes are stored offsite. 
The AHC-IS servers provide a stable, secure, well-maintained, and high-capacity data 
storage environment, and both REDCap and MySQL are widely-used, powerful, reliable, 
well-supported systems. Access to the study's data in REDCap will be restricted to the 
members of the study team by username and password. 
Division of Epidemiology and Community Health, School of Public Health, University 
of Minnesota 
(Primary location Dr. Lisa Harnack, Co-I) 

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Nutrition Coordinating Center: 
The Nutrition Coordinating Center (NCC) is located in the Division of Epidemiology and 
Community Health, and occupies about 6,000 square feet of office space. Dr. Lisa 
Harnack (Co-I) is the Director of the NCC since 2007. The NCC includes office space for 
staff, telephone calling stations, resource area, training room, and conference rooms. 
The NCC has developed and maintains a research-quality food and nutrient database 
linked to computerized interactive interview software, the Nutrition Data System for 
Research (NDSR). The NDSR is designed primarily for clinical research and 
epidemiological studies investigating relationships between diet and health.  The NDSR 
dietary data collection and nutrient analysis tool is well recognized in the nutrition 
research community and has been licensed for use in hundreds of research studies. 
Clients may also choose to enlist the services of NCC to accomplish some or all of their 
dietary data collection and processing objectives. The NCC Service Center offers 
training and certification of dietary interviewers, collection of dietary intake by 
telephone interview, processing of food records, analysis of menu and recipe data, 
development of dietary data collection protocols, and customized support services for 
additional related research needs. The Service Center has provided the aforementioned 
types of services to hundreds of studies since 1974, including carrying out entry and 
nutrient analysis of food records obtained via meal observation procedures similar to 
those to be utilized in the proposed study. 
The Nutrition Coordinating Center was established in 1974, and continues to address 
the ongoing needs of the nutrition research community. The Center is located on West 
Bank of the University of Minnesota campus and is a 10 minute drive from Dr. Chow’s 
office. 
Department of Biochemistry, Molecular Biology and Biophysics. 
(Dr. Doug Mashek, Co-I) 
Office: 
 Dr. Mashek’s laboratory is located on the University of Minnesota Molecular Cell 
Biology Building which is connect by walkway to Dr. Chow’s office and the CRU (5 
minutes walk). Dr. Mashek’s office is 125 ft2 and the laboratory has office space within 
the laboratory for 9 people.  
Computer:  
Dr. Mashek has a new iMac that is equipped with all the needed software (Microsoft 
Office, Photoshop, etc.). Additionally, he also has a Macbook Air laptop and the 
laboratory has 7 Dell desktops.  
Laboratory: 

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Dr. Mashek’s lab is approximately 1600 ft2 plus two adjacent 125 ft2 rooms for tissue 
culture and radioisotope studies. The laboratory is equipped with a tissue culture hood, 
two fume hoods, an autoclave and a walk-in cold room. The following equipment are 
either in Dr. Mashek’s laboratory or is readily accessible: Class 2, Type A2 biological 
safety cabinet, NuAire, Inc.; water jacketed CO2 incubator, NuAire, Inc.; Nucleofector II, 
Amaxa Biosystems, Inc.; Synergy HT multi-detection microplate reader, BIO-TEK 
Instruments, Inc.; FLUOstar OPTIMA microplate reader, BMG Labtech; Mini-Protean 3 
Cell and Trans-Blot electrophoretic transfer Cell, BIO-RAD; PowerPac HC Power Supply, 
BIO-RAD; CKX41SF2 inverted microscope, Olympus; IX70 inverted microscope with 
fluorescence and imaging capabilities, Olympus; Labphoto 2 microscope, Nikon; Saturn 
5 GC/MS, Varian; PTC-200 DNA Engine Cycler, BIO-RAD; ABI StepOne Plus Real-Time 
PCR system, Applied Biosystems; SMARTSPEC Plus, BIO-RAD; Spinchron R refrigerated 
centrifuge, Beckman Instruments; RT6000B refrigerated centrifuge, Sorvall; J2-21 
centrifuge, Beckman; Orion Star Series pH meter, Thermo Electron; L8-70 
Ultracentrifuge, Beckman; Ultralow temperature freezer, Sanyo; AQT series top loading 
balance, Adams Equipment; A30 analytical balance, Mettler, Inc.; G24 environmental 
incubator shaker, New Brunswick Scientific Co., Inc; Gyrotory water bath shaker-model 
G76, New Brunswick Scientific Co., Inc.; general purpose water bath, Precision, 
Seahorse XF-96, Seahorse Bioscience. 
Relevant Resources Available to Dr. Mashek 
Center for Mass Spectrometry and Proteomics (CMSP) 
This facility contains diverse instrumentation capable of generating different data types 
for workflow development, including newly installed Thermo Fisher Fusion Orbitrap 
mass spectrometry for high sensitivity proteomics analysis, along with a Proxeon 
nanoLC system; A newly installed AB Sciex 5600+, along with Eksigent nanoLC for 
targeted, SWATH analyses and other proteomic operating modes; A newly installed Q-
Exactive instrument (Thermo) equipped with Dionex LC capable of both nanocapillary 
and high flow is a main instrument for high resolution metabolomics analysis. 
Complementing this instrumentation is a Thermo LTQ-Velos mass spectrometer 
equipped with Eksigent nanoLC and ETD ionization and a Sciex/AB Qtrap 5500 both 
equipped with fully automated nanoflow LC systems. In addition to this 
instrumentation, the facility also houses ten additional mass spectrometers offering full 
services for small (metabolites) and large (proteins) molecule biological mass 
spectrometry applications. The facility also has a Leco GC-GC TOF-MS instrument for 
small molecule/metabolite studies, and also a Waters LCT Premiere TOF-MS for 
metabolomic profiling. 
Department of Pediatrics 
(Dr. Justin Ryder Co-I). 

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Computer and Information Technology Resources 
Dr. Ryder has a personal desktop computer and a laptop computer including necessary 
software (Microsoft Office, Reference Manager, SPSS, SAS, R, GraphPad, Adobe 
Acrobat Pro), to use for analysis of data, manuscript preparation, and any other 
activities associated with the conduct of the study.  Each member of Dr. Ryder’s study 
team has a personal computer capable of performing all necessary activities associated 
with the study.  Dr. Ryder has full access to the wide-array of computer services offered 
at the University of Minnesota.  These include network infrastructure, web-access, 
email, etc.  The Department of Pediatrics is supported by the Academic Health Center 
Information Services, which provides comprehensive support for computer, hardware, 
and software. Dr. Ryder’s office is a 5 min walk from Dr. Chow’s office. 
Office Facilities and Supplies 
Fully equipped office facilities and supplies are available to Dr. Ryder and his staff 
within the Department of Pediatrics and the CTSI.  The Division of Epidemiology and 
Clinical Research has approximately 5,000 square feet of office space located in the 
Moos Health Sciences Tower, which is located in close proximity to the CTSI and 
hospital.  These facilities include desk space, filing cabinets, telephones, network 
copiers, scanners, fax machines, printers, and a postage meter.   
Relevant Resources Available to Dr. Ryder 
Center for Magnetic Resonance Research 
Dr. Ryder will also utilize the Center for Magnetic Resonance Research (CMRR for 
measurement of liver fat content via MRI collected during the study. The CMRR is fully 
furnished and equipped for modern translational and clinical research. The facility 
house multiple ultrahigh magnetic fields (7 Tesla and above) but also contains three 3 
Tesla (Siemens, Berlin, Germany) scanners which will be used in this study. The 3 Tesla 
Trio (Siemens) system is a standard system similar to those used in hospitals for clinical 
diagnosis. This system can be used for standard imaging or spectroscopy 
measurements of liver fat. The CMRR is equipped with extensive computation 
resources, including onsite file servers and compute servers are located in a modern 
secured server room with diesel-backed UPS power and fully redundant cooling. The 
center also includes subject rooms for consenting participants, or other participant 
needs. The center provides MR Technologists to run the 3T scanners, assist with data 
collection, and maintain equipment and troubleshoot scanner issues. This location is a 
10 minute walk from Dr. Chow’s office and the CRU. 

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Coordinating Centers for Biometric Research (CCBR) and Biostatistical Design & 
Analysis Center (BDAC) 
(Dr. Erika Helgeson, Co-I, Qi Wang) 
Communications, Photocopying, Faxing, Electronic Images 
Dr. Helgeson’s office is located in the CCBR, which is part of the Division of Biostatistics 
in the School of Public Health at the University of Minnesota. The CCBR is connected to 
the internet via fiber-optic cable to the University network backbone which provides 
internet access for all staff members. The CCBR also has 8 fax modems for receiving 
images of case report forms, surveys, and other hard-copy documents. Case report 
forms received this way are automatically filed into patient folders. In addition, the 
CCBR also has two high-speed copying machines capable of collating, 2-sided copying, 
reduction and stapling. These two multi-function machines also serve as high-speed 
printers and as high-speed scanners for electronic imaging needs. 
Computer Servers 
Several SPARC servers (Solaris operating system) and Intel servers (Linux and FreeBSD 
operating systems) provide application service to the CCBR and the Division of 
Biostatistics. Access to these applications is provided by more than 100 thin-client (X-
servers) desktops. All servers are connected to the Internet. The CCBR machines have 
more than 20 terabytes of disk storage available. Statistical analysis and data 
management are the primary applications provided by the servers. Statistical analyses 
are performed using SAS, SAS/GRAPH, SAS/IML, and R. Oracle is the primary relational 
database management system. Various SQL clients are used including SAS, Perl, 
SQL*Plus, and PHP. 
Ms. Wang has an office at BDAC, which is a sub-unit of the University's Clinical and 
Translational Science Institute (CTSI).  She has a current Windows machine with access 
to the internet through the University's Academic Health Center's IS system, with all 
standard software (e.g., the Microsoft Office Suite) as well as SAS and R.  
Salk Institute 
The Salk Institute for Biological Studies 
Environment. The Salk Institute offers an excellent intellectual environment for the 
scientific success of its faculty. Dr. Panda’s lab is part of the Regulatory Biology 
Laboratories and is housed in the same floor as the Laboratory of Genetics. The open 

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lab structure of the institute offers constant scientific exchange among researchers and 
PIs. Open lab design of the Salk Institute fosters persistent interactions among these 
lab members. Panda lab has ongoing collaborations with several Salk Institute faculty in 
the field of metabolism, endocrinology, and data science. They include Ron Evans, Marc 
Montminy, Reuben Shaw, Saket Navlakha, Alan Saghatelian with whom Panda lab has 
several active collaboration projects. These colleagues and their scientific staffs offer a 
productive intellectual environment relevant to the success of this project. 
Laboratory. Laboratory space for 10 full time researchers furnished with standard 
laboratory equipment (thermocyclers, benchtop centrifuges, dissecting microscopes, 
DNA/RNA, protein gel electrophoresis apparatus, heat blocks, water baths, freezers, 
refrigerators, tissue culture hoods, tissue culture incubators, Q-PCR, FPLC) is available 
within the department of Regulatory biology at Salk institute. 
Clinical. The lab has developed, validated, and deployed the myCircadianClock app for 
monitoring and intervening human eating pattern. The app is hosted on secured HIPAA 
compliant Amazon Web Server (AWS). The database and the backend processes 
communicate with the user’s smartphone using double encryption. 
Computer. The laboratory has 12 recent model PCs, and 3 Macintosh computers. In 
addition, shared facilities at Salk include high-end personal computers for image 
processing, database searches, and modeling. Salk institute’s central Research 
Computing department maintains an extensive data communications network available 
to all laboratories. 
Office. The PI has a 100 sq. ft. office located adjacent to the laboratory; desk with 
computer are available for staff associated with the project. 
Other. The Salk Institute has several service cores including microarray, transgenic, 
machine shop, metabolic, rodent behavior, microscopy, antibody production, peptide 
synthesis, and deep-sequencing cores. These cores are run by expert technicians and 
directors who constantly help and train lab personnel in carrying out their experiments. 
Equipment – The Salk Institute for Biological Studies 
Panda lab occupies ~2000 sqft space in the regulatory biology section of the Salk 
institute. The lab has wetlab are and dry lab area. This collaboration relates to the dry 
lab area of Panda lab. 

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Dry-lab. 
The lab has a dedicated ~550 sqft space for dry lab computation area that houses 
personnel, computer and equipment for data collected through digital devices and 
clinical measurements. The dry-lab area contains 5 desktop computers with updated 
software for analyses of diet, physical activity, sleep, and survey data collected 
through relevant wearable devices, smartphones, paper- or computer-generated 
questionnaire. 
The lab has ~120 wrist-worn activity monitors for use in human studies for longitudinal 
monitoring of sleep, activity and light exposure. 
The dry-lab area has secure physical storage space for storing back-up hard drives, 
paper copies of informed consent documents, clinical lab reports, and other human 
subject related information. 
Through Salk Institute’s high-speed internet access line the lab has access to HIPAA 
compliant cloud server. 
The lab has a Tanita body composition scale, and other necessary tools for collecting 
anthropometric measurements from human subjects. 
Facilities: Colorado 
Dr. Bergman 
The facilities and other resources available to the research team include 
everything needed to complete the proposed studies. Dr. Bergman has been 
performing human research studies similar to the one proposed for 18 years at this 
institution, so the infrastructure and collaborations are ready to successfully complete 
this project. In addition to Dr. Bergman’s laboratory, there is an NIH funded Clinical 
Translational Research Center (CTRC) at the University of Colorado Anschutz Medical 
campus (UCAMC) for all human work proposed. The intellectual environment is filled 
with other NIH funded investigators who are performing work complimentary to what 
is proposed, and there is a strong history of human clinical research and funding at our 
institution. Together, these facilities available to the research team provide a scientific 
environment that strongly supports the proposed research and, therefore, collectively 
contributes to the success of the project. 
Dr. Bergman has approximately 1000 sq. ft. of wet laboratory space located in 
the School of Medicine to support the proposed research. He has all the necessary 
equipment and expertise to perform muscle dissection, western analyses, cell culture 
experiments, and tissue incubation required for these analyses as they are standard 
procedures in the laboratory. Specifically, he has a fume hood, an Organomation 100 
position multivap nitrogen evaporator, Beckman clinical centrifuge, a Sanyo -80C  
freezer, 2 stand up freezers, 2 refrigerators, Mettler-Toledo analytical balance, pH 
meter, gel rocker, BioRad power supplies and western equipment. Additionally, Dr. 

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Bergman has half of a 120 sq. ft cold room, and a 200sq. ft. cell culture laboratory 
across the hall from his laboratory containing two incubators, an inverted microscope, 
water bath, refrigerator, centrifuge, and a cell culture hood.  Dr. Bergman’s GC/MS and 
LC/MS instruments are on the 6th floor of the building containing his laboratory, in a 
space designated as core space for the Colorado Nutrition Obesity Research Center. 
Importantly, he has all the necessary internal standards and bone fide standards 
required to identify and quantify neutral lipids including diacylglycerol isomers, polar 
lipids such as acyl-carnitines, as well as deoxysphingolipids via LC/MS. These analyses 
are routine in the Bergman laboratory.   
 
HOW THE ENVIRONMENT WILL CONTRIBUTE TO SUCCESS 
We are an integrated, interdisciplinary team with unique expertise to complete the 
proposed study. Dr. Chow has experience recruiting and conducting studies in 
overweight participants from her ongoing NIH grant (R01DK098203) which trained 
sedentary overweight/obese participants for 16 weeks. The proposed study will 
leverage this existing infrastructure for recruitment and implementation (screening, 
consent, metabolic assessment, training). Dr. Mashek (Co-I on current R01, 4 papers 
with Dr. Chow) will analyze the acquired serum, lend his expertise on hepatic fat 
metabolism/hepatic insulin resistance on the observed findings, and have regular 
discussions with Dr. Chow about interpreting the findings within the current scientific 
literature. Dr. Ryder (3 papers with Dr. Chow) will provide his expertise in measuring 
body fat using MRI, supported by Dr. Pat Bolan. Dr. Dengel has an established working 
relationship with Dr. Chow (Co-I on current R01, 8 joint publications) and will lend his 
expertise in on the indirect calorimetry and DXA measurements. Dr. Harnack is an 
internationally recognized nutritional epidemiologist and registered dietician. She will 
provide her expertise in nutritional assessment (1 submitted R01 with Dr. Chow as MPI) 
and will work with Dr. Chow in documenting the caloric intake and co-supervising the 
study dietician to ensure compliance with the intervention. Dr. Satchinananda Panda 
(Co-I with Dr. Chow on current HFHL grant) will lend his expertise in time restricted 
eating (TRE) and provide support for the myCircadianClock (mCC) app. Dr. Bergman 
(Director of Molecular and Cellular Analytical Core Facility [Colorado 
NORC:P30DK48520] will perform the stable isotope analysis from the plasma. Dr. Erika 
Helgeson (1 submitted R01 with Dr. Chow) and Qi Wang (5 papers with Dr. Chow) will 
provide the statistical support for the proposal.  
Adequate personnel are in place to support the proposed study. For the study, Dr. 
Chow has her own research group (one physician assistant, one study coordinator) and 
can draw upon the research staff from the University of Minnesota Division of Diabetes 
and Endocrinology with clinical trial expertise (1 physician assistant, 3 research nurses 
and 3 study coordinators) if needed. Dr. Chow will work with Dr. Harnack to leverage 
the existing infrastructure of the NCC to develop and implement the meal observation 
protocol.   

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The team will have scheduled meetings to ensure study progression as shown Table 1.  
Table1: Meeting Schedule during Study 
Personnel 
Role 
Schedule  
(May meet more frequently if needed) 
Dr. Harnack and 
Research study staff  
Study recruitment, flow 
and progress 
Weekly in-person 
Dr. Mashek 
Review scientific literature 
relevant to study, 
measure serum 
inflammatory markers 
Weekly in-person (Dr. Chow already 
attends) 
Dr. Panda’s team 
Review participant 
compliance and 
adherence via review 
using the mCC app 
Weekly by phone or email 
Dr. Panda 
Review study progress 
Every 6 months by phone 
Dr. Dengel and Dr. 
Ryder 
Address issues related to 
indirect calorimetry 
measurement (Dr. Dengel) 
and MRI (Dr. Ryder) 
Every 2 months in-person 
Dr. Helgeson and Qi 
Wang 
Organize study setup and 
data flow; review study 
progress; data analysis 
Bi-weekly for 3 months in-person, then 
every 6 months while study is ongoing, 
bi-weekly for last 6 months of grant 
Dr. Bergman 
Analysis of blood samples 
for stable isotope 
(glucose, glycerol) 
enrichment 
Monthly for 3 months by phone to set 
up analysis procedures, q6 months 
during sample acquisition phase, then 
q3 months during sample analysis 
phase 
Dr. Moheet – Patient 
Safety Officer 
Follow up adverse events 
Every 6 months in-person and prn 
event occurrence 
 
Of note, the University of Minnesota CTSI received a CTSA award in 2018 to support the 
clinical research. Adequate personnel and infrastructure are in place to support the 
proposed clinical study 
 
 

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