Effect of Ultra Processed Versus Unprocessed Diets on Energy Intake
Phase: NA
Conditions: Healthy Diet
Sponsor: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)

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Clinical Research Protocol
National Institute of Diabetes and Digestive and Kidney Diseases
Date: August 17, 2020
PROTOCOL NUMBER: 18-DK-0044
PROTOCOL VERSION: 1.6
TITLE: Effect of Ultra Processed versus Unprocessed Diets on Energy Intake
SHORT TITLE: Processed Food and Energy Intake
IDENTIFYING WORDS: Energy intake, processed foods
TYPE OF PROTOCOL: Natural History – Disease Progression/Physiology
START DATE: September 2017
END DATE: February 2021
PRINCIPAL INVESTIGATOR:
Kevin Hall, Ph.D.*
Laboratory of Biological Modeling (LBM)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Building 12A, Room 4007
12 South Drive, (MSC 5621)
Bethesda, MD 20892-5621
Phone: 301-402-8248
Fax: 301-402-0535
E-mail: kevinh@niddk.nih.gov

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Table of Contents
Précis................................................................................................................................................9
1. Introduction and Background................................................................................................10
2. Study Objectives........................................................................................................................10
2.1  Primary Aim: ....................................................................................................................11
2.2  Exploratory Aims: ............................................................................................................11
3.1  Subjects..............................................................................................................................12
3.2  Inclusion and Exclusion Criteria.....................................................................................12
3.3 Study Schedule...................................................................................................................13
3.3.1 Screening Procedures....................................................................................................13
3.3.2 Study Timeline..............................................................................................................14
4. Analytical Procedures................................................................................................................16
4.1 Resting energy expenditure ..............................................................................................16
4.2 Respiratory chamber.........................................................................................................16
4.3 24-hour urine collection ....................................................................................................17
4.4 Doubly Labeled Water (DLW).........................................................................................17
4.5 Plasma substrate and hormone concentrations ..............................................................17
4.6 Questionnaires....................................................................................................................18
4.7  Dual energy x-ray absorptiometry (DXA)......................................................................20
4.8 Physical activity monitoring .............................................................................................20
4.9 Oral glucose tolerance test ................................................................................................20
4.10 Continuous Glucose Monitoring (CGM).......................................................................21
4.11 Sleep Monitor...................................................................................................................21
4.12 MRI/MRS .........................................................................................................................21
4.13 Saliva Collection...............................................................................................................21
4.14 Psychophysical Taste Task: Sucrose and Salt Detection Thresholds .........................22
4.15 Psychophysical Taste Task: Sucrose and Salt Preference ...........................................22
5.  Statistical Analysis....................................................................................................................23
6.  Safety Considerations ...............................................................................................................24
6.1 Possible Risks and Hazards ..............................................................................................24
6.2 Risks Related to Clinical Relevance of Test Results.......................................................26
6.3 NIH Reporting Requirement............................................................................................26
6.3.1 Adverse Event Definitions............................................................................................27
6.3.2 Expedited Reporting .....................................................................................................27

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6.3.3 IRB Requirements for PI Reporting at Continuing Review .........................................27
6.3.4 Clinical Director Reporting ..........................................................................................27
6.4 Data and Safety Monitoring Plan.....................................................................................27
7.  Recruitment Strategies..............................................................................................................28
7.1 Recruitment of Women, Children, Minorities and Other Vulnerable Individuals.....28
8.  Subject Withdrawal ..................................................................................................................29
9.  Research Use, Storage, and Disposition of Human Subjects’ Samples and Data ....................29
9.1 Collaborations Using Stored Samples..............................................................................30
10.  Informed Consent ...................................................................................................................30
11.  Risk/Benefits to Study Participants ........................................................................................31
12.  Remuneration..........................................................................................................................31
13. References................................................................................................................................33
14. Appendix A, Proposed Schedule .............................................................................................36

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Précis
Overconsumption of foods that result from extensive industrial processing is believed to 
contribute to the development of obesity. Ultra-processed foods now represent most of the calories 
consumed in America and their contribution to overall diet has increased in parallel with the rise 
in the prevalence of obesity over the past several decades. While such correlations are suggestive, 
the effect of industrial processing per se, independent of dietary macronutrient composition, on ad 
libitum energy intake has not been carefully investigated. Therefore, we will conduct feeding 
studies in  adult men and women to investigate the differences in ad libitum energy intake resulting 
from consuming test diets for a pair of 2-week periods in a randomized, crossover design during a 
single 4-week period. The test diets presented to participants will be matched for calories, 
macronutrient composition, sugar, fiber, and sodium, but one diet will be composed of ultra-
processed foods whereas the other diet will be unprocessed foods. 
1. Introduction and Background
The rise in prevalence of obesity and type 2 diabetes over the past several decades has been 
mirrored by an increasingly industrialized food system [1] involving substantial refinement of 
inexpensive “inputs” (primarily corn, soy, and wheat) to generate a seemingly diverse supply of 
convenient, stable, inexpensive, “added value” foods [2, 3] with supernormal appetitive properties 
[4, 5]. Ultra-processed foods [6] have becoming increasingly common worldwide [7] and now 
contribute to the majority of calories consumed in America [8]. 
Overconsumption of ultra-processed foods has been implicated as a causative factor in weight 
gain [9] and recommendations to reduce intake of processed foods are shared by a variety of 
proposed healthy dietary approaches that are widely divergent from a nutrient perspective [10]. 
Whereas previous studies have examined the effect of single meals varying in their degree of 
industrial processing on satiety and glycemia [11], no study has yet examined the effect of 
industrial processing on long-term ad libitum energy intake. 
2. Study Objectives
In this study, we will investigate ad libitum food intake of 20 healthy adult volunteers who will 
each complete a 4-week stay at the NIH. During their stay, they will consume an ultra-processed 
and an unprocessed diet for 2 weeks each, in random order. We will collect information (primary 
and exploratory endpoints detailed below) about energy intake and metabolic changes, in response 
to the two test diets. These test diets that will be presented to subjects in amounts exceeding their 
daily energy requirements; subjects will be instructed to eat as much or as little of each diet as 
desired.
The diets will be designed using a standard computerized nutrition database, and foods will be 
selected using a food classification system that categorizes foods as belonging to one of three 
groups: unprocessed or minimally processed foods, processed culinary or food industry ingredients, 
or ultra-processed food products [6]. Diets will maximize differences in industrial processing while 
matching for calories, macronutrient composition, sugar, fiber, and sodium. Each subject will be 
admitted as an inpatient to the NIH Clinical Center and will stay on the 5SWN Metabolic Care 
Research Unit (MCRU).

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Previous studies have successfully detected significant effects of modifying dietary fat 
versus carbohydrate on ad libitum energy intake over periods of 1-2 weeks [12, 13], We 
hypothesize that the degree of industrial processing will also significantly impact ad libitum 
energy intake even if the presented diets are matched for calories, macronutrients, sugar, sodium, 
and fiber. 
Hypothesis: The hypothesis of this study is that an ultra-processed diet will lead to greater mean 
ad libitum energy intake as compared to an unprocessed diet when the meals presented to the 
subjects are matched for daily calories, macronutrient composition, sugar, fiber, and sodium.
2.1  Primary Aim:  
1. To determine differences in ad libitum energy intake (kcals) during 2 weeks of eating an 
ultra-processed diet as compared to 2 weeks of an unprocessed diet matched for 
presented calories, macronutrient composition, sugar, fiber, and sodium.
Previous studies in similar numbers of subjects have shown changes in insulin sensitivity after 2-
4 weeks of consuming diets that differ in advanced glycation end products that are produced 
through high heat cooking typical of industrially ultra-processed foods [14, 15]. One study 
suggested that consumption of meals composed of processed foods results in lower energy 
expenditure compared to consuming unprocessed foods [16]. Hepatic triglyceride content can 
also be altered with isocaloric diet changes in as little as 9 days [17]. Therefore, we will explore 
changes in glucose tolerance, various inflammatory markers, energy expenditure, and body 
composition metrics as follows:    
2.2  Exploratory Aims: 
1. To determine changes in oral glucose tolerance after 2 weeks of consuming an ultra-
processed diet as compared to 2 weeks of consuming an unprocessed diet matched for 
presented calories, macronutrient composition, sugar, fiber, and sodium. 
2. To determine differences in immune system function and inflammatory markers (c-
reactive protein, E-selectin, soluble intercellular adhesion molecule-1, soluble vascular 
adhesion molecule-1, serum amyloid A, tumor necrosis factor-α, and interleukins 6 and 
8) after 2 weeks of consuming an ultra-processed diet as compared to 2 weeks of 
consuming an unprocessed diet matched for presented calories, macronutrient 
composition, sugar, fiber, and sodium. 
3. To determine changes in body weight and body composition after 2 weeks of consuming 
an unprocessed diet matched for presented calories, macronutrient composition, sugar, 
fiber, and sodium.
4. To determine changes in energy expenditure and substrate metabolism after 2 weeks of 
consuming an ultra-processed diet as compared to 2 weeks of consuming an unprocessed 
diet matched for presented calories, macronutrient composition, sugar, fiber, and sodium.
5. To determine changes in hepatic triglyceride content after 2 weeks of consuming an ultra-
processed diet as compared to 2 weeks of consuming an unprocessed diet matched for 
presented calories, macronutrient composition, sugar, fiber, and sodium.
2.3 Second Cohort
The first cohort of 20 subjects completed this study in late 2018 and 
the ultra-processed diet significantly increased energy intake by 508 kcal/d (p=0.0001) compared 

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to the unprocessed diet – an effect that was not significantly related to sex or BMI. The increased 
energy intake was due to excess consumption of dietary carbohydrate and fat, but protein intake 
was stable between the diets. 
The hypothesized mechanisms for the effect of the ultra-processed diet on energy intake 
include:
i)
The non-beverage energy density of the meals (calories per unit mass) was higher 
during the ultra-processed versus the unprocessed diet (1.96 kcal/g versus 1.06 
kcal/g, respectfully). Therefore, more calories were consumed for a similar mass of 
food eaten;
ii)
The ultra-processed meals were consumed more rapidly which may have delayed 
satiety; and
iii)
The protein content of the ultra-processed diet was very slightly lower than the 
unprocessed diet (14% versus 15.6% of total calories, respectively) which may have 
led to increased overall energy intake to match absolute protein intake according to 
the protein leverage hypothesis.
We propose enrolling a second cohort of 20 subjects who will be exposed to a reformulated 
ultra-processed diet that more closely matches the unprocessed diet regarding non-beverage 
energy density and protein content. The ultra-processed diet will continue to be matched in terms 
of daily calories, macronutrient composition, sugar, fiber, and sodium. The ultra-processed diet 
will be modified to include foods that are typically eaten more slowly. If these factors were the 
main reasons that original ultra-processed diet led to the observed ~500 kcal/d increase in energy 
intake over the unprocessed diet, then the reformulated ultra-processed diet should lead to a 
smaller difference in energy intake between diets. Except for dietary formulation, the study 
design, procedures, and primary and exploratory aims, will be identical in the 2 cohorts. We will 
also compare the differences in ad libitum energy intake between ultra-processed and 
unprocessed diets between  cohorts 1 and 2.
3.  Study Design
This is a randomized order crossover study of healthy adult volunteers to determine 
differences in ad libitum energy intake when consuming an ultra-processed vs. unprocessed diet 
matched for presented calories, macronutrient composition, sugar, fiber, and sodium.
3.1  Subjects
Adult subjects (18-50 years of age) will be recruited to the study via the NIH Patient 
Recruitment and Public Liaison Office.  Telephone pre-screening will exclude subjects with 
diabetes or any other metabolic disorders, and those who require assistance to complete activities 
of daily living. All others will be contacted by the protocol team to review exclusion criteria.  All 
subjects will be fully informed of the aims, nature, risks, and potential benefits of the study prior 
to giving written consent. 
3.2  Inclusion and Exclusion Criteria
Inclusion criteria:  
1. Adults age 18-50 years, male and female
2. Weight stable (< ± 5 % over past 6 months)

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3. Body mass index (BMI) ≥ 18 kg/m2 
4. Willing to cease their habitual caffeine intake during the study, beginning one week prior 
to inpatient admission
5. Written informed consent
6. Willing to eat the food provided in the study 
7. Able to complete daily bouts of stationary cycling at a moderate rate and intensity with a 
HR equal to or greater than 0.3×(220-age-HRrest)+HRrest but not exceeding 0.4×(220-age-
HRrest)+HRrest and no signs of arrhythmia
Exclusion criteria:
1. Evidence of metabolic or cardiovascular disease, or disease that may influence metabolism 
(e.g. cancer, diabetes, thyroid disease) 
2. Taking any prescription medication or other drug that may influence metabolism (e.g. 
diet/weight-loss medication, asthma medication, blood pressure medication, psychiatric 
medications, corticosteroids, or other medications at the discretion of the PI and/or study 
team)
3. Hematocrit < 34% (women only)
4. Hematocrit < 40% (men only)
5. Pregnancy, lactation (women only)
6. Participating in a regular exercise program (> 2h/week of vigorous activity)
7. Caffeine consumption > 300 mg/day 
8. Regular use of alcohol (> 2 drinks per day), tobacco (smoking or chewing) amphetamines, 
cocaine, heroin, or marijuana over past 6 months
9. Eating disorders and psychological conditions such as (but not limited to) claustrophobia, 
clinical depression, bipolar disorders, that would be incompatible with safe and successful 
participation in this study, as determined by investigators.
a.
Past or present history of claustrophobia since part of the protocol will involve 
being confined to a small room for whole-body indirect calorimetry and being in 
an MRI scanner for liver fat measures
10. Implants, devices, or foreign objects implanted in the body that interfere with the Magnetic 
Resonance procedures
11. Volunteers with strict dietary concerns (e.g. vegetarian or kosher diet, food allergies)
12. Volunteers unwilling or unable to give informed consent
13. Non-English speakers due to unavailability of required questionnaires in other languages
3.3 Study Schedule
3.3.1 Screening Procedures
The Principal Investigator or Associate Investigator will discuss the nature of the study and 
answer any questions the subject may have. All subjects will be fully informed of the aims, 
nature, risks, and potential benefits of the study prior to giving written informed consent.
Potentially eligible volunteers will be invited to the NIH Clinical Center for a screening visit 
that will include a medical history and physical examination and other screening procedures.  
Subjects will be required to fast for at least 12 hours before the first screening visit, which will 
include a blood draw for assessment of blood lipid profile, liver panel, electrolytes, and blood 
count.  The blood draw will also be used to screen for the presence of diabetes, which will be 
defined as a fasting glucose > 126 mg/dl or %HbA1c > 6.5%. Female subjects of reproductive 

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potential will complete a urine pregnancy test during this visit, and positive test results will 
preclude subjects from further participation. 
All subjects who are interested in participating in the full study protocol will have the following 
procedures and tests performed: 

Medical history (including food allergies or intolerances) and physical examination 

Resting electrocardiogram (EKG)

Resting energy expenditure (REE)

Fasting blood tests

Body weight and height

Submaximal Exercise EKG

One 20-minute stationary bicycling session with predetermined speed and resistance 
identified using the results from the resting and exercise EKG tests.

Questionnaires
o
Food Frequency Questionnaires 
o
MacArthur Socio-Economic Status Questionnaire
o
Three-Factor Eating Questionnaire
o
Profile of Mood States Questionnaire
o
Sleep Apnea Clinical Score (SACS)
o
STOP-Bang Questionnaire
o
DSM-5 Self-Rated Level 1 Cross-Cutting Symptom Measure [18]
o
Owl and Lark Sleep Questionnaire (may also be completed during inpatient 
admission)
The goal of the submaximal exercise EKG is to achieve and maintain a target heart rate (HR) 
equal to or greater than 0.3×(220-age-HRrest)+HRrest but not exceeding 0. 4×(220-age-
HRrest)+HRrest and show no signs of arrhythmia. The 20-minute stationary cycling bout in the 
prescribed range will be used to assess ability to perform a minimal amount of exercise and also 
to determine and practice the speed and intensity for the inpatient admission. This speed and 
intensity will be repeated on days of scheduled cycling exercise during the inpatient visit. Any 
clinically significant findings from the screening procedures will be communicated to the subject 
by a qualified member of the research team and appropriate follow-up with their primary care 
physician will be planned. The food frequency questionnaires (FFQ) will be analyzed to help 
identify any dietary restrictions or food avoidances as well as to ensure that the subject’s habitual 
diet meets the inclusion/exclusion criteria.
Volunteers will be asked to sample the diets during this visit. They will eat foods from the 
Ultra Processed and Unprocessed menus supervised on the metabolic unit. If qualified for entry 
into the study based on the inclusion/exclusion criteria and the study team’s assessment of their 
likelihood of adherence to study procedures, study staff will arrange for active participation in 
the study as soon as the next day.
3.3.2 Study Timeline
Up to 20 healthy volunteers will be studied as inpatients to the MCRU. The outline of the 
study is shown in Figure 1. Every effort will be made to adhere to the proposed timelines, but 
some flexibility is required for scheduling of other studies, unanticipated equipment 

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maintenance, etc. Scheduling variations will not be reported. For a calendar overview of the 
proposed timeline, please see Appendix A.
Figure 1. Study outline.
Subjects will complete the following procedures during the study period:

Fasting blood draw

Doubly labeled water dosing

Resting energy expenditure (measured by indirect calorimetry using a bedside metabolic 
cart)

Body fat by dual energy X-ray absorptiometry (DXA; whole body)

Metabolic weight and height

Liver fat content by magnetic resonance spectroscopy

24-hour respiratory chamber  

Oral glucose tolerance test (OGTT)
All subjects will have daily body weight measurements and will wear accelerometers 
throughout the study except during bathing. Subjects will also be required to perform exercise on 
a stationary bicycle at a constant pace and intensity (as determined at the screening visit) for a 
total of 60 minutes (recommended to occur in three 20 minute blocks) every day during their 
inpatient stay. Subjects will spend one 24-hour period each week residing in a respiratory 
chamber and will have liver fat content measured towards the end of each diet period. A baseline 
DXA scan will be obtained, followed by weekly DXA scans for a total of 5 scans. Similarly, the 
indirect calorimetry measurements will be obtained at baseline and on a weekly basis. An oral 
glucose tolerance test (OGTT) will be performed at the end of each diet period. During the study 
period, female subjects of reproductive potential will also complete weekly urine pregnancy tests 
prior to DXA scans, and positive test results will preclude subjects from further participation. 
Participants will consume 3 meals per day in the metabolic unit. Meals will be ad libitum and 
consumed in the metabolic unit. After meal times, all meal trays will be checked, and uneaten food 
will be weighed.  Dietitians and research staff will meet with the subjects regularly to discuss the 
diet and assess compliance. Subjects will be provided with snacks corresponding to the prevailing 
diet assignment and will keep a record of all foods consumed outside the meal times.
Subjects will receive one of two test diets with 7-day rotating menus: an ultra-processed foods 
diet or an unprocessed foods diet. The meals presented to subjects will be matched for calories and 
macronutrient content. The daily macronutrient composition of both test diets will be 
approximately 47% carbohydrate, 36% fat and 17% protein. See Table 1 for an example of a daily 

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menu for each diet. All subjects assigned to the same diet will receive identical meals (e.g., see 
Table 1) with instructions to eat as little or as much food as desired. Meals will be delivered 3 
times per day and subjects will be given 60 minutes to finish their meal. Snacks will also be 
provided every morning so that subjects have access to snacks as desired throughout the day. The 
food provided to the subjects will substantially exceed their estimated energy requirements 
(approximately 200% of energy requirements as determined by 1.6×REE measured at screening) 
and will likely be more than the subjects can eat. Participants will also be provided with bottled 
water to consume throughout the day and the amount of water consumed will be recorded. 
Table 1.  Examples of unprocessed and ultra-processed diets 
Unprocessed Diet
Ultra Processed Diet
Breakfast
Greek yogurt parfait with 
strawberries, blueberries and granola 
with walnuts
Banana
Pancakes with margarine and syrup
Turkey sausage
Tater tots
Apple juice
Lunch
Spinach salad with chicken, apples, 
bulgur, sunflower seeds and grapes
Vinaigrette
Turkey sandwich with American cheese and 
mayo on white bread
Baked potato chips
Diet ginger ale
Dinner
Beef tender roast
Rice pilaf (basmati rice with garlic, 
onions and peppers)
Steamed broccoli
Side salad with balsamic vinaigrette
Oranges
Apple slices
Pecans
Cheeseburger
French fries and ketchup
Diet ginger ale
Greek yogurt
Canned peaches in heavy syrup
4. Analytical Procedures
4.1 Resting energy expenditure
Resting energy expenditure (REE) will be measured in the morning with the subject in a supine 
position. Subjects will be instructed to complete a 12-hour overnight fast prior to each scheduled 
REE. An indirect calorimeter with the ventilated hood technique will be used for the approximately 
30-40 min measurement, where the first 5-15 minutes will be excluded from analysis.  The 
respiratory quotient will be calculated as the ratio of carbon dioxide production to oxygen 
consumption.  Resting metabolic rate will be calculated using the principles of indirect calorimetry 
using the VO2 and VCO2 measurements [19]. 
4.2 Respiratory chamber
Subjects will be in the chamber for a total of 2 days during each diet treatment period. The 
respiratory chamber is a specially constructed room to assess the metabolism of subjects for a 
period of 24 hours. Designed as a walk-in “pull” calorimeter, it is an open circuit unit that draws 

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conditioned room air into the chamber at the same flow rate as it is extracted into the gas analysis 
system.  Each of the rooms is equipped with a toilet and sink with privacy screen, cycle 
ergometer, bed, desk, and computer with access to television and other forms of entertainment. 
Food and beverages are passed through an air-lock drawer system.  Electrocardiogram telemetry 
and a nurse call are available to enhance subject safety.  In the chamber, 24h energy expenditure 
(EE), sleeping EE, respiratory quotient (RQ), and the thermic effect of feeding (TEF) will be 
assessed. 
4.3 24-hour urine collection
During each 24-hour respiratory chamber stay, all urine will be collected for measurement 
of total nitrogen, creatinine, catecholamines, and C-peptide.
4.4 Doubly Labeled Water (DLW)
On the morning beginning diet treatments 1 and 2, we will collect two baseline (predose) urine 
samples. These baseline urine samples will be collected prior to DLW dose. Each DLW dose will 
be obtained from a larger DLW batch containing a mixture of 2H2O (99% enrichment) and H218O 
(10% enrichment). The dose of DLW will be prepared individually for each subject (proposed 
dosage: 1 g DLW/kg of body weight). 
After the baseline urine samples are collected, DLW will then be given to the subject to drink. 
After the subject has consumed the DLW, two rinses (each rinse is about 50 mL of tap water) will 
be added to the dosing container, and given to the subject to drink.  Spot urine samples will be 
collected periodically during the subsequent 6 to 12 hours of the DLW dose administration and 
daily thereafter. The exact time of urine collection for each sample will be recorded. Isotopic 
enrichments of urine samples will be measured by isotope ratio mass spectrometry for the next 13 
days. The average CO2 production rate will be estimated from the differential disappearance of the 
2 isotopes in the urine samples [20].  The energy expenditure calculations will use the average 24 
hr RQ measurements from the respiratory chamber. 
4.5 Plasma substrate and hormone concentrations
Blood samples will be collected into chilled test tubes containing preservatives.  Those 
samples for which analysis of gastrointestinal peptides will take place will also contain a 
dipeptidyl peptidase IV inhibitor and a protease inhibitor (e.g., 4-(2-aminoethyl)benzenesulfonyl 
fluoride) and be collected into chilled glass tubes.  All samples will be kept on ice and then 
centrifuged for isolation of plasma.  After centrifugation, the plasma will immediately be frozen 
and stored for later analysis.  Our current plans for measuring plasma substrate and hormone 
concentrations are described in the procedures outlined in Table 2.  
Table 2. Example list of procedures for measuring plasma substrates and hormones
Metabolite/hormone
Proposed Method
Metabolites
Triglycerides
Fatty acids
Cholesterol
Glucose
Chem15
colorimetric assay
colorimetric assay
colorimetric assay
colorimetric assay
Hemoglobin A1C
Hormones
Insulin
RIA

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Leptin
C-peptide
Adiponectin
GLP-1 (active form)
PYY3-36
GIP
Acyl- and total ghrelin
FGF21
TSH
Triiodothyronine (T3, f-T3)
Thyroxine (T4, f-T4)
Reverse-T3
RIA
RIA
RIA
RIA
RIA
RIA
RIA
ELISA
CLIA
CLIA
CLIA
ELISA
Markers of
Inflammation
CRP
IL-6
IL-8
TNF-alpha
E-selectin
sICAM-1
sVCAM-1
serum amyloid A
ELISA
ELISA
ELISA
ELISA
ELISA
FMIA
FMIA
ELISA
RIA = radioimmunoassay; HPLC = high performance liquid chromatography, ELISA = enzyme-
linked immunosorbent assay; CLIA = chemiluminescence immunoassay; FMIA = fluorescent 
multiplexed bead-based assay
4.6 Questionnaires
Profile of Mood States (POMS) Questionnaire:  The Profile of Mood States (POMS) is a widely 
used questionnaire for measuring distinct mood states. The POMS has 65 items, each with a 5-
point adjective rating scale, which measures six identifiable moods or feelings: Tension-Anxiety 
(T), Depression-Dejection (D), Anger-Hostility (A), Vigor-Activity (V), Fatigue-Inertia (F) and 
Confusion-Bewilderment (C). Completion of the POMS takes 5-10 minutes.
DSM-5 Self-Rated Level 1 Cross-Cutting Symptom Measure:  This questionnaire is delivered 
during the screening visit and assesses mental health domains that are important across 
psychiatric diagnoses. It consists of 23 questions that assess 13 psychiatric domains, including:

Depression

Anger

Mania

Anxiety

Somatic symptoms

Suicidal ideation

Psychosis

Sleep problems

Memory

Repetitive thoughts and behaviors

Dissociation

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
Personality functioning

Substance use
Each item asks about how much (or how often) the individual has been bothered by the specific 
symptom during the past 2 weeks. Each item on the measure is rated on a 5-point scale. Cutoff 
scores, critical items, and follow-up measures are provided for each disorder.  The questionnaire 
will be scored by a qualified member of the study team immediately after completion.  If the 
subject scores above the cutoff for one or more disorders, these results will be discussed with a 
medically qualified team member, and a Clinical Center psychiatry consult will be obtained as 
appropriate.
Food Frequency Questionnaires (FFQ):  Food Frequency Questionnaires (FFQ) are self-
assessment questionnaires developed to identify foods and macronutrients that significantly 
contribute to energy intake [21]. Subjects will be given instructions to complete a form, which 
will be reviewed for completeness by clinic staff and any blank answers will be resolved. The 
FFQ contains questions on the frequency and portion size of consumption of certain food items 
over a defined period of time.
Three-Factor Eating Questionnaire:  The three-factor eating questionnaire (TEFQ) is a self-
assessment questionnaire developed to measure dietary restraint, disinhibition and hunger [22].  
The questionnaire contains 36 items with a yes/no response, 14 items with a 1-4 response scale 
and 1 item with a 1-5 response scale. 
MacArthur Socioeconomic Status (SES) Questionnaire:  The MacArthur SES questionnaire is a 
widely used self-assessment questionnaire.  It begins with subjective social status questions, 
followed by questions assessing educational attainment, occupational status, income and assets.
Owl and Lark Sleep Questionnaire:  The Owl and Lark Sleep Questionnaire is a self-assessment 
questionnaire to determine morningness and eveningness in human circadian rhythms. The 
questionnaire contains 19 questions that require the subject to score based on wake and sleep 
habits. 
Sleep Apnea Clinical Score (SACS): This questionnaire contains four items: measurement of 
neck circumference, history of hypertension, and two reported clinical symptoms (e.g. snoring, 
gasping during sleep). With these criteria, a sleep apnea clinical score (SACS) is generated, and a 
score greater than 15 indicates high risk for sleep apnea. 
STOP-Bang Questionnaire: This questionnaire contains eight items, four of which are yes/no 
questions about snoring, tiredness, observed breathing during sleep, and blood pressure. The 
other four items are cut-off physical measurements and demographics (BMI, age, neck 
circumference, gender). Answering yes to three or more items indicates a high risk of OSA. 
Hunger & Satiety Assessment:  At several time points throughout the study, subjects will be 
asked to complete a survey to identify their perception of hunger (i.e., visual analog scale [VAS]) 
[22].  More specifically, the VAS survey will consist of four questions: 1) "How hungry do you 
feel right now?" 2) "How full do you feel right now?" 3) "How much do you want to eat right 
now?" and 4) "How much do you think you can eat right now?"  Below each question on the 

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survey there is a horizontal 100mm line with qualifying statements such as “Not at all”/“The 
least I can possibly” and “Extremely”/“The most I can possibly”, anchoring the line on the 
extreme left and right side, respectively.  In response to each question, subjects will be asked to 
draw a vertical mark on the horizontal line to represent the magnitude of their response to the 
question.  A value for each response is quantified by measuring the distance of their mark (in 
mm) relative to the left end of the line.  Therefore, the values (or "scores") for each question 
range from 0 to 100. On a few select days, data will be collected at least every 30 to 60 minutes 
over the next 2-3 hours after consuming each of their meals. It will take approximately 3 minutes 
for a participant to complete the assessment at a given time.
Sensory and palatability assessments: On several days throughout the study, subjects will be 
asked to complete a survey to assess the palatability and sensory properties (such as taste, 
texture, etc.) of the meals provided. Subjects will complete 100-point VAS line scale ratings of a 
series of measures, including meal liking and selected sensory properties. Survey items will be 
completed before eating, after the first bite, and after the participant has finished eating. Ratings 
include: how filling they expect a meal to be (expected fullness), how quickly or slowly they felt 
they ate the meal, and the extent to which they feel hungry, full and thirsty, their desire to eat and 
prospective consumption. The scale responses range from “Not at all” (0) to “Very” (100), 
except for prospective consumption, which is anchored by “Nothing at all” (0) and “The most I 
have ever eaten” (100). These ratings will be embedded amongst distracter “mood” ratings of 
alert, happy, and clear-headed. It will take approximately 3 minutes for a participant to complete 
the assessment at a given time.
4.7  Dual energy x-ray absorptiometry (DXA)
DXA scans will be performed at baseline followed by weekly scans using the General Electric 
Lunar iDXA (General Electric; Milwaukee, WI, USA) to determine body composition. 
Participants will change into a hospital gown, remove all metal-containing objects and lie on a 
table while the scanner, which emits low energy X-rays, passes along the body. The scan will take 
approximately 10 minutes. The radiation dose per scan is less than 1 mrem, and therefore the total 
of 5 scans planned per subject is below the guideline of 5000 mrem per year allowed for research 
subjects by the NIH Radiation Safety Committee. This is an FDA approved device that is being 
used as per approved labeling and parameters. 
4.8 Physical activity monitoring
Physical activity will be quantified daily with an activity monitor using high sampling 
frequencies during all waking periods. Overall physical activity levels, daily changes, amount of 
time spent in sedentary, moderate, vigorous intensity categories and activity-associated energy 
expenditures will be extracted [23]. This device is an FDA cleared Class II medical device within 
the United States and is being used per approved labeling and parameters.
4.9 Oral glucose tolerance test
After an overnight fast, 75 grams of oral glucose will be administered. Blood samples will 
be obtained at 0, 10, 20, 30, 60, 90, 120, and 180 minutes to measure glucose and insulin 
concentrations. Blood samples for research will also be collected. 

15
4.10 Continuous Glucose Monitoring (CGM)
 Subjects will wear an FDA-approved CGM during their inpatient admission except 
during the MRS procedure. The device will be used to record glucose in real-time, approximately 
every 5 minutes. The system consists of a small sensor, transmitter, and hand-held receiver 
(about the size of a pager). The small sensor, with a small needle attached, will be inserted 
subcutaneously. The transmitter, which is attached to the sensor, will send the measured glucose 
to the receiver. The data stored in the receiver will be uploaded to a software for analysis. The 
sensor is changed at least once every two weeks. This is an FDA approved device that is being 
used as per approved labeling and parameters. 
4.11 Sleep Monitor
The sleep scanner is a wearable tracking device intended for monitoring sleep patterns, 
which may affect metabolism. The sleep scanner collects data on respiratory effort, oxygen 
saturation, pulse, snoring, body position, and movement. This device consists of a sensor board, 
a respiratory belt (respiratory impedance plethysmography), and oximetry probe. Subjects will 
apply the respiratory belt around the diaphragm and oximetry probe clips to their finger. Subjects 
will be asked to apply the device at bedtime for several nights during the study period. The 
wearable tracking device is synced to a computer and data will be retrieved for analysis. 
4.12 MRI/MRS
The subject will lie supine in the MR gantry and will be asked to hold their breath during 
scans.  The duration will depend on their tolerance. The MR imaging test will be performed in 
collaboration with another NIH investigator, Dr. Ahmed Gharib. Subjects will undergo the 
MRI/MRS after signing a separate informed consent document specific to the imaging study, 
protocol 11-DK-168 (Dr. Gharib’s study). This separate imaging study consent also involves CT 
scan. A CT scan of your heart may be performed to confirm findings from our MRI study. However, 
the CT scan portion is optional and refusal to undergo CT scan will in no way affect participation 
in the current study.  MR spectroscopy will be performed primarily for assessment of hepatic fat 
content. Each MRI scan will last no more than 2 hours.  
Gadolinium, a contrast agent sometimes administered to enhance visualization of the heart and 
blood vessels, will not be used during MR study scans. The MRI used in this study is FDA 
approved and it is being used as per approved labeling and parameters. 
4.13 Saliva Collection
Variation in basic taste receptor genes (sweet, salty, sour, bitter, and umami) and nutrient 
sensing genes may contribute to differences in food preferences and dietary patterns. The taste 
receptor genes and their variants will be chosen accordingly for sweet and umami (T1R [taste 
receptor, type 1]), bitter (T2R [taste receptor, type 2]), and salty (ENaC [epithelial sodium 
channel][24]). The saliva sample will be collected using Orangene® (Genotek, Kanata, Canada) 
and used for the isolation of genomic DNA. Genomic DNA samples will be used as a template in 
TaqMan® assays (Applied Biosystems, Foster City, CA) in duplicates.  Genotyping will be done 
by investigators blind to any subject information. Samples will be stored in a -80 °C freezer. For 
genotype quality assurance, the concentration of DNA in stored samples will be checked using a 
Nanodrop® Spectrophometer ND100 prior to use. The observed genotypes will be checked for 
Hardy–Weinberg equilibrium. The exploratory analysis will investigate the relationship of taste 
and obesity-related genotype with dietary intake, and with sweet and salt preference and 

16
thresholds. An analysis of variance will test whether taste detection thresholds or preference 
differ by genotype. Saliva collection takes approximately 2 minutes to complete. This procedure 
will be conducted at the beginning of the study period.
4.14 Psychophysical Taste Task: Sucrose and Salt Detection Thresholds
Sucrose and salt detection thresholds will be assessed using a two-alternative forced-
choice staircase procedure was developed at the Monell Center for Adults [25-27]. The two-
alternative forced choice is a psychophysical method developed to elicit responses about an 
individual’s experience regarding a stimulus. It focuses on the evaluation of a single attribute 
(e.g., sweetness or saltiness), and the stimulus is adjusted based on the individual’s responses 
[28, 29]. For this study, all testing will take place in a private, comfortable room. Subjects will be 
fasted for at least one hour before the task and acclimate to the testing room and to the researcher 
for approximately 15 minutes before testing.  For the first trial and each subsequent trial, subjects 
will be presented with pairs of solutions in random order; within each pair, one solution will be 
distilled water, and the other will be the taste stimulus. Subjects will be instructed to taste the 
first solution presented within the pair, swish the solution in their mouth for 5 seconds, and 
expectorate. This will be repeated for the second solution within the pair. Between solutions, 
subjects will rinse their mouth with water; they will rinse once within a pair, and twice between 
successive pairs. After tasting both solutions within a pair, subjects will be asked to point to the 
solution that has a non-neutral taste. A tracking grid will be used to record subjects’ responses 
[30-32].  This method eliminates the need for a verbal response and has been shown to be an 
effective method for assessing both taste and olfaction in children [30, 33]. The task takes 
approximately 30 minutes to complete. Participants will complete this task at the end of each test 
diet period.
4.15 Psychophysical Taste Task: Sucrose and Salt Preference
Sucrose and salt preference will be assessed using a two-series paired comparison-
tracking method developed at the Monell Center for Adults [25-27]. Subjects will be presented 
with pairs of solutions differing in sucrose concentration (3, 6, 12, 24, and 36 g per 100 mL) and 
salt (0.92–6.14% wt/vol NaCl). They will be asked to taste the solutions without swallowing and 
point to which of the pair they liked better. Subsequently, each pair presented will be determined 
by the subject’s preceding preference choice. The entire task is then repeated with the stimulus 
pairs presented in reverse order. After completion of the taste task, the geometric mean of the 
sucrose and salt concentrations chosen will be determined. This serves as an estimate of the 
participant's most preferred level of salt or sucrose [33, 34]. The task takes approximately 15 
minutes to complete. Participants will complete this task at the end of each test diet period.
4.16 Above-threshold or Suprathreshold sensory function
Subjects will be trained on the use of the general labelled magnitude scale (gLMS) 
before we measure perceived intensities. Two trials consisting of 4 ascending concentrations of 
each stimulus (sucrose, NaCl) with the first “concentration” being water will be presented to 
the subjects. All four concentrations will be presented in random order without repeat. We will 
use 0.00, 0.09, 0.35, and 1.05 mol/l sucrose solutions and 0.00, 0.16, 0.38, and 1.05 mol/l salt 
solutions. Subjects will rate the perceived intensity of the stimulus using the gLMS scale and 
mean intensities of the two trials at each concentration for each stimulus to evaluate subjects’ 
taste intensity perception.

17
The task takes approximately 10 minutes to complete. Participants will complete this task at the 
end of each test diet period.
4.17 Slips-of Action Paradigm
Goal-directed behaviors are guided by the assessment and valuation of possible outcomes. 
Thus, if the outcome of a certain behavior is devalued, the behavior is less likely to be performed. 
Habit responding occurs when an individual’s actions are driven by external stimuli, which trigger 
and automatic behavioral response. This task assesses such learning in three stages: control 
discrimination training, congruent discrimination training, and an incongruent conflict task. In the 
initial stage if subjects respond correctly to the cue box instructions they will receive a reward. 
For the second stage, no feedback will be provided for correct or incorrect responses. This stage 
of the task should be controlled predominantly by a goal-directed system.  The third stage 
features a conflict between the previously associated cues and outcomes. The total task time will 
be 27 minutes. Participants may complete this computer based task during the inpatient 
admission, depending on time.
4.18 Reward Prediction Error
A computerized probabilistic procedural learning task, the Probabilistic Selection Task, to 
assess participants ability to learn from both positive and negative outcomes. Dopamine plays a 
key role in reinforcement learning. Learning from positive outcomes is thought to be a result of 
phasic bursts firing of dopamine which stimulates the D1R direct (“Go”) pathway and inhibits the 
D2R indirect pathway (“Stop”). The dip in dopaminergic firing experienced as a result of negative 
outcomes is thought to support learning to avoid the unrewarded choice. This task trains 
participants on reward contingencies and then tests their learning strategy to determine whether 
they are more adept at learning predominantly from positive   feedback (suggestive of high phasic 
dopamine response) or from negative outcomes (suggestive of sufficient degree of dip in phasic 
and tonic dopamine response). Both phases of the task combined last approximately 25 minutes. 
Participants may complete this computer based task during the inpatient admission, depending on 
time.  
5.  Statistical Analysis
Day to day variability of ad libitum energy intake has a standard deviation of about 500-
600 kcal/d [35-37]. Therefore, over a 14-day diet period each subject will have a mean energy 
intake with a standard error of about 130-160 kcal/d and the mean energy intake difference 
between the study diets will have a standard error of about 190-230 kcal/d. Using the conservative 
assumption that within-subject energy intake correlations are zero, the 20-subject study cohort will 
have an 80% power to detect a difference in mean ad libitum energy intake between the diets of 
about 125-150 kcal/d with a Type I error probability of 0.05. 
Previous ad libitum diet studies comparing mean energy intake between 14 day periods 
consuming diets differing in macronutrient content have observed differences >250 kcal/d [12, 13] 
which is greater than the minimum detectable difference for a 10-subject cohort under the same 
assumptions as above. Therefore, the 20-subject cohort of our study is adequately powered to 
detect physiologically significant differences in ad libitum mean energy intake between the diets.

18
6.  Safety Considerations
6.1 Possible Risks and Hazards
Research-related risks in this study include those associated with study procedures, 
namely blood drawing, indirect calorimetry, respiratory chamber, doubly labeled water, 
measurement of body composition by dual energy x-ray absorptiometry (DXA), wearing 
physical activity monitors, continuous glucose monitoring, sleep monitoring, oral glucose 
tolerance testing, and measurement of liver fat by MRS.  There are no study medications, and we 
do not anticipate any diet intervention-related risks since the study will be under close 
supervision with trained dietitians/nutrition experts.  
Blood drawing.  The placement of intravenous needles may cause transient pain, and may also 
result in bruising, bleeding, and/or clotting at the site of needle insertion. A medical provider will 
be available should any of these problems occur. There is a possibility that a catheter placement 
would be unsuccessful or need to be removed.  If this should occur, another catheter would be 
placed. The approximate volume of blood to be drawn for research purposes is as follows:
Research Blood Draws
Screening Visit
Diet 1
Diet 2
Total
10 ml
174 ml
147 
331
This is within the NIH guidelines for limits of blood drawn for research purposes (550 ml in any 
8-week period).  
Oral Glucose Tolerance Test. Subjects may experience faintness, nausea, and vomiting after 
taking the glucose solution.
Indirect calorimetry.  The use of the ventilation hood may cause some minimal discomfort in 
claustrophobic subjects. 
DXA.  The amount of radiation during the DXA scan is less than one mrem to the whole body.  
This radiation exposure is below the guideline of 5000 mrem per year allowed for research 
subjects by the NIH Radiation Safety Committee The use of the DXA scan apparatus may cause 
some minimal discomfort in claustrophobic subjects and may cause some minimal back pain in a 
small minority of the individuals. 
Doubly labeled water.  The doubly labeled water procedure has no known risks. 
Respiratory chamber.  Besides inconveniences that can reasonably be expected as a result of 
spending an extensive time (24h) in the live-in room calorimeter, there is no risk to subjects’ 
physical health. Claustrophobia is an exclusionary criterion.  All subjects will be given an 
opportunity to experience the metabolic chamber prior to enrollment in the study. 

19
Profile of mood states questionnaire. There is no known risk associated with the POMS 
questionnaire, however, there is the potential of subjects finding completing the task tedious or 
some may find the questions probing and too personal in nature to comfortably answer.  Subjects 
will be informed that they do not have to respond to all the questions if they have reservations 
about sharing such personal information.
DSM-5 Self-Rated Level 1 Cross-Cutting Symptom Measure. There is no known risk associated 
with this self-report measure. However, there is the potential of discovering clinically relevant 
information requiring further follow-up. If this is the case, a member of the research team will 
contact the subject, and appropriate follow up will be planned. 
Food frequency questionnaire.  There is no known risk associated with the FFQ.
Three-factor eating questionnaire.  There is no known risk associated with the TFEQ.
MacArthur Socioeconomic Status (SES) Questionnaire.  There are no known risks associated 
with the MacArthur SES questionnaire although some subjects may find completing the task 
tedious or some may find the questions probing and too personal in nature to comfortably 
answer.  Subjects will be informed that they do not have to respond to all the questions if they 
have reservations about sharing such personal information.
Owl and Lark Sleep Questionnaire.  There is no known risk associated with the sleep self-
assessment. 
Hunger and satiety assessment. There are no known risks associated with the hunger and satiety 
assessment
Sleep Apnea Clinical Score. There is no known risk associated with the Sleep Apnea Clinical 
Score. 
STOP-Bang Questionnaire. There is no known risk associated with the STOP-Bang 
Questionnaire.
Sensory and palatability assessments. There are no known risks associated with the sensory and 
palatability assessment.
Physical activity monitors.  There are no risks associated with the monitors, but subjects may 
find them to occasionally be inconvenient. 
Continuous Glucose Monitoring System. According to the device manufacturer, there is minimal 
risk associated with the device. Possible side effects include but are not limited to local infection, 
inflammation, pain or discomfort, bleeding at the insertion site, bruising, itching. Because this 
protocol represents the first use of this device by our research team, we do not know the 
frequency of such complications of the device. A medical provider will be available should any 
of these problems occur. 

20
Sleep Monitor. Subjects may experience discomfort from the respiratory belt or oximetry probe. 
A plastic covering of the respiratory belt sensor board safeguards against direct skin contact with 
electrical parts and wiring. Participants will wear the device over thin clothing.
MRI/MRS. There is a small chance of claustrophobia or muscle-skeletal discomfort from lying 
partially in the magnet. During the imaging measurement, the noise may be somewhat 
unpleasant, but ear plugs will be provided for comfort. Although the long-term risk of exposure 
to a magnetic field is not known, the possibility of any long-term risk is extremely low from the 
information accumulated over the past ten years.
MRS Warning:  Certain implants, devices, or foreign objects implanted in the human body 
may interfere with the MR procedure.  Volunteers who have undergone specific prior 
surgeries (i.e. heart, brain, gastric bypass, breast augmentation, etc) and/or have implants 
of specific types may be required to provide their IMPLANT CARD in order to determine 
implant safety/compatibility with the magnet before a scan is performed. The continuous 
glucose monitor will be removed prior to the MRS procedure.
Saliva collection for taste genotyping. This is a non-invasive procedure and there are no risks 
associated with saliva collection. Information regarding genetic results will not be shared with 
the participants. 
Psychophysical taste task: Sucrose and Salt Detection Thresholds. There are no risks associated 
with assessments of taste detection thresholds.
Psychophysical taste task: Sucrose and Salt Preference. There are no risks associated with 
assessments of taste preference.
Above-threshold or Suprathreshold sensory function:  There are no risks associated with 
assessments of taste preference.
Slips of Action Paradigm:  There are no risks associated with the Slip-of-Action paradigm 
assessment.
Reward prediction error: There are no risks associated with the Reward Prediction Error 
assessment.
6.2 Risks Related to Clinical Relevance of Test Results
If any lab tests, questionnaires or any other measurements made during the screening or 
procedures of this protocol show clinically significant abnormalities that may impact the health 
and well-being of the subjects, they will be notified by a qualified member of the research team. 
Appropriate follow-up with their primary care physician will be planned. If needed, the research 
team will refer subjects to a health care provider. 
6.3 NIH Reporting Requirement
6.3.1 Adverse Event Definitions
Please refer to definitions provided in Policy 801: Reporting Research Events.

21
6.3.2 Expedited Reporting
Please refer to the reporting requirements in Policy 801: Reporting Research Events.
6.3.3 IRB Requirements for PI Reporting at Continuing Review
Please refer to the reporting requirements in Policy 801: Reporting Research Events.
6.3.4 Clinical Director Reporting
Problems expeditiously reported to the IRB in iRIS will also be reported to the Clinical Director. 
A separate submission is not necessary as reports in iRIS will be available to the Clinical 
Director.
6.4 Data and Safety Monitoring Plan
Given the modest level of risk involved in the research, no Data and Safety Monitoring 
Board will be instituted. Medical oversight will be provided by the Medically Accountable 
Investigator. Adverse events will be recorded and monitored by the Principal Investigator. The 
study will be subject to audits to ensure compliance with the protocol and applicable regulatory 
requirements consistent with the NIDDK Monitoring plan. Audit results will be reported to the 
Principal Investigator for further reporting as appropriate. Study documents and pertinent 
hospital or clinical records will be reviewed to verify that the conduct of the study is consistent 
with the protocol plan. 
7.  Recruitment Strategies
Adult subjects will be recruited from the community through advertisements in 
clinicaltrials.gov and possibly, local flyers, newspapers, magazines, internet, mail, radio, or 
television. 
The age range for inclusion in this protocol is 18-50 years.  The rationale for excluding subjects 
older than 50 years is multi-fold.  First, a number of the major metabolic outcome variables of this 
protocol have been shown to change significantly with aging (e.g., metabolic rate and body 
composition).  Second, much of the current information on our primary outcome measurements 
was gathered from subjects within the age range specified by this protocol; therefore, we believe 
that this age range is appropriate as it allows us to put our results into direct context with the current 
knowledge base. 
7.1 Recruitment of Women, Children, Minorities and Other Vulnerable Individuals
NIH employees will be eligible to participate in this study. We will use appropriate methods to 
recruit staff participants, posting flyers where public announcements are permitted (rather than 
direct solicitation of subordinates). Staff subject’s data will be kept private and confidential. We 
will provide the NIH Information Sheet on Staff Research Participation to staff prior to obtaining 
consent to help them understand the possible consequences of their participation. Neither 
participation nor refusal to participate as a research subject will have an effect, either beneficial or 
adverse, on the staff participant’s employment or position at NIH. Additional safeguards may be 
employed, including independent consent monitoring, training study staff on how to 
obtain/manage potentially sensitive information about a co-worker.  

22
We will actively encourage the participation of women and minorities.  We expect women to 
represent approximately half of the study population, and we anticipate that African-Americans 
and Hispanics will constitute at least 20% of enrollees.
We have deliberately chosen not to study children and adolescents at this time. At present, there 
is no information suggesting that the metabolic responses to alterations of dietary macronutrients 
are likely different in adults and children and so we see no compelling reason to expose children 
to the rigors and inconvenience of this type of study.  We fully recognize, however, that the insights 
derived from the data analysis in this study may very well direct our attention to other potentially 
fruitful areas of clinical research involving child health. We have also chosen not to enroll pregnant 
women at this time, as there is no known prospect of direct benefit for the woman or the fetus, and 
we see no reason to expose either the woman or the fetus to the rigors and inconvenience of this 
type of study.
Individuals who are unable to provide consent will also not be enrolled. This is based on 
the strenuous demands of the study and the requirement to be able to read, understand and carefully 
follow study directions, and fill out several questionnaires throughout the study. We have chosen 
not to enroll non-English speaking participants, as these questionnaires are not currently available 
in languages other than English. We will make every effort to enroll minority men and women. 
8.  Subject Withdrawal
The subject may choose not to be in the study, or, if they agree to be in the study, they may 
withdraw from the study at any time. If a subject withdraws from the study, no new data will be 
collected for study purposes unless the data concern an adverse event related to the study.  If such 
an adverse event occurs, we may need to review the subjects’ entire medical record. Subjects’ 
decision not to participate or to withdraw from the study will not involve any penalty or loss of 
benefits to which they are entitled, and will not affect access to health care at the National Institutes 
of Health.
Also, there are several conditions that require the Principal/Co-Investigator to drop a study 
volunteer from this protocol, which include but are not limited to the following:

Development of any new medical condition or start of medications that would have 
prevented enrollment in this study as it pertains to the exclusion criteria

Inability or unwillingness to comply with study requirements

The subject becomes pregnant during the course of the study

The Principal/Co-Investigator of the study site deems it unsafe to remain in the study

The study is terminated
9.  Research Use, Storage, and Disposition of Human Subjects’ Samples and 
Data
As with all clinical data, the findings will be kept confidential. Volunteer clinical data will be 
protected and tracked using standard operating procedures in the medical record department.  All 
research charts and records will be kept in a secure place in a locked file cabinet in the office of 
the Principal Investigator.  All research samples and data will be identified by a study code linked 
to the subject’s name and the code and the results of all analyses will be kept strictly confidential.  
All research samples (e.g., blood) will be coded for storage in refrigerators and freezers in a locked 

23
NIH laboratories. These samples will be stored indefinitely.  The IRB will be notified in the event 
these samples are accidentally destroyed, lost or are anonymized.
Some clinically relevant research data will be stored indefinitely in the medical record and will 
be accessible to the subject for review by others of the participant’s choosing (doctors, insurance 
companies etc.) after executing a release of information.  This and other data will be maintained 
in databases, password protected and secure.
9.1 Collaborations Using Stored Samples
All stored samples and data that will be sent to collaborator/s will be stripped of subjects’ 
personal identifiable information based on NIH guidelines. Any future collaborations requiring 
transfer of stored biological samples and data will be coded after informing the IRB and obtaining 
necessary assurances from the outside institution. The protocol will be amended and IRB approval 
will be sought when such collaborations are established. The following are the current 
collaborations on the study: 

Dr. Ciarán Forde at the National University of Singapore will receive data from sensory 
and palatability assessments of the diets.

Dr. Michelle Cardel at the University of Florida will receive data from questionnaires and 
assessments about the diets as well as energy intake data to look at the effects of 
objective and subjectively measured social status and potential relationships to energy 
intake and diet quality.  

Dr. Tera Fazzino at the University of Kansas will receive data from questionnaires and 
assessments about the diets as well as energy intake data to test whether palatability may 
provide additional predictive utility for energy intake.

Dr. Jeff Brunstrom at the University of Bristol of the United Kingdom will receive data 
regarding energy intake and macronutrient profile of meals to analyze whether 
differences in intake can be partially explained by the specific beverages in each meal 
and their role in modifying the overall energy density of the items consumed.

Multiple collaborators at NCI will: (i) identify candidate biomarkers that discriminate 
between an ultra-processed and an unprocessed diet, (ii) identify a metabolomic profile of 
an ultra-processed diet, and (iii) identify candidate biomarkers of common UPF items and 
ingredients, such as dietary emulsifiers and high-fructose corn syrup. 

Multiple collaborators at NIAID will characterize effects on immune phenotype measured at 
the following levels:
o
 Transcriptome using PBMC, or preferably, whole blood sampled in PAXgene tubes. 
o
 PBMC phenotype or phosphorylation responses, using mass cytometry measuring ~40 
antigens.  
o
Serum/plasma proteomics using SomaLogic to measure 1300 proteins. 

24
10.  Informed Consent
Written informed consent will be obtained from the participant prior to any screening visits, 
study procedures or treatments. The Principal Investigator or other designated qualified protocol 
investigators will explain the study in language understandable to the subject. Sufficient time and 
opportunity will be given for discussion of the research as well as to answer any questions they 
may have, taking care to minimize or eliminate the perception of coercion or undue influence. The 
participant and the investigator will sign the current IRB-approved informed consent document.  
A copy of the consent will be given to the subject for future reference. The signed documents will 
be sent to the Medical Records Department for placement in the subject's permanent CC medical 
record. The consent process will additionally be documented in the electronic medical record 
(CRIS). 
 
11.  Risk/Benefits to Study Participants
11.1 Known Potential Risks
Please refer to Section 6 “Safety Considerations” for the list of possible risks/hazards. 
11.2 Known Potential Benefits
There will be no other direct benefits from participation in this study aside from the 
knowledge that they are contributing to advancing our understanding of obesity, and that these 
insights may lead to new treatment options in the future. Abnormal values will be discussed 
with the study volunteers and forwarded to their primary care physicians with the subject’s 
permission.
11.3 Assessment of Potential Risks and Benefits
Please refer to section 6 of the protocol.  
12.  Remuneration
Subjects will receive payment for the time and effort connected with the study according to the 
table below. Completion of the entire study needs to be strongly encouraged since incomplete data 
collection provides no scientific benefit despite introducing a small amount of risk to the subject. 
Therefore, we propose the following remuneration schedule: 
•
Total Reimbursement* for completing both diet treatments according to the table below:
* If early discharge occurs prior to study completion, total reimbursement will be at the rate of 
$40.00 per day, to include the day of discharge.  This reimbursement rate goes into effect if the 
study is not completed for any reason, i.e. occurrence of illness, family emergency, or per 
investigator discretion in the event of noncompliance with study procedures.  On a case-by-case 

25
basis, adjustments may be made to the study schedule, including repeat testing to accommodate 
subjects who must leave the study temporarily in the event of an emergency.
The total remuneration for this study, if completed, is in accord with our previous study that 
included the same number of study days (09-DK-0081; $6520) and a similar level of 
inconvenience for the volunteers.
Procedure
P
n
TOTAL
Daily Food Adherence
$60.00
28
$1,680.00
Inpatient per Diem
$40.00
28
$1,120.00
Body Composition (DXA)
$50.00
5
$250.00
Indirect Calorimetry with cart 
$50.00
5
$250.00
Daily Weight and Accelerometer (per week) 
$50.00
4
$200.00
Oral Glucose Tolerance Test (OGTT)
$100.00
2
$200.00
Doubly Labeled Water (DLW)
$50.00
2
$100.00
Questionnaires
$100.00
1
$100.00
24hr Respiratory Chamber
$100.00
4
$400.00
MRS for Liver Fat
$100.00
3
$300.00
Continuous Glucose Monitoring (per week)
$100.00
4
$400.00
Sleep Monitoring
$50.00
4
$200.00
 
TOTAL 
REMUNERATION:
$5,200.00

26
13. References
1.
Stuckler, D., et al., Manufacturing epidemics: the role of global producers in increased 
consumption of unhealthy commodities including processed foods, alcohol, and tobacco. 
PLoS Med, 2012. 9(6): p. e1001235.
2.
Blatt, H., America's food: What you don't know about what you eat. 2008, Cambridge: The 
MIT press. 336.
3.
Roberts, P., The end of food. 2008: Houghton Mifflin Harcourt Publishing Company. 400.
4.
Kessler, D.A., The end of overeating: controling the insatiable American appetite. 2009: 
Rodale Inc. 320.
5.
Moss, M., Salt, sugar, fat: how the food giants hooked us. 2013: Random House. 446.
6.
Monteiro, C.A., et al., A new classification of foods based on the extent and purpose of 
their processing. Cad Saude Publica, 2010. 26(11): p. 2039-49.
7.
Monteiro, C.A., et al., Ultra-processed products are becoming dominant in the global food 
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29
14. Appendix A, Proposed Schedule
1 MRS times: Mon PM, Tues PM, Thurs PM
2 If needed
Date 
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
 
Processed/Unprocessed Diet
Unprocessed/Processed Diet
24 h energy expenditure 
(chamber)
X
X
X
X
24 h urine collection
X
X
X
X
Liver fat content (MRS)1
X
X
X
DXA scan
X
X
X
X
X
REE/metabolic cart
X2
X
X
X
X
Fasted blood draw
X
X
X
Doubly labeled water dose
X
X
DLW urine sample(s)
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Body weight
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Accelerometer
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Plex sleep monitor 
X
X
X
X
X
X
Oral glucose tolerance test 
(OGTT)
X
X
Hunger & satiety assessments
X
X
X
X
X
X
CGM sensor 
insertion/replacement
X
X
X
X
Pregnancy test (females)
X2
X
X
X
X
Sensory & palatability 
assessments
X
X
X
X
Sucrose & salt detection
X
X
Sucrose & salt preference
X
X
Saliva collection
X