Novel Approach to Assess Metabolic Flexibility in a Respiratory Chamber
Phase: NA
Conditions: Diabetes Mellitus, Type 2
Sponsor: Pennington Biomedical Research Center

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Novel Approach to Assess Metabolic 
Flexibility in a Respiratory Chamber 
“METFLEX” 
 
 
 
 
 
  
 
 
 
 
 
 
PROTOCOL 
 
 
 
 
 
 
 
 
 
 
Principal Investigator: David McDougal, PhD 
Co-Investigator: Kara L. Marlatt, PhD, MPH  
Co-Investigator: Eric Ravussin, PhD 
Co-Investigator: Leanne Redman, PhD 
Medical Investigator: Frank Greenway, MD 
 
 
 
 

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1. 
BACKGROUND AND SIGNIFICANCE 
Metabolic flexibility is the ability of an organism—as a whole, or at the organ, tissue, or 
cellular level—to adjust fuel oxidation to fuel availability. It was originally described as “the 
capacity to switch from lipid oxidation during fasting conditions to the suppression of lipid 
oxidation and subsequent increase glucose uptake, oxidation, and storage under insulin-
stimulated conditions” [1]. During the non-physiological, highly-controlled conditions of a 
hyperinsulinemic-euglycemic clamp, individuals with insulin resistance and type 2 diabetes 
are less metabolically flexible to carbohydrate (CHO), and therefore, have an impaired 
capacity to increase muscle and whole-body glucose oxidation and storage that is reflected 
by a blunted increase in respiratory quotient (RQ). However, Galgani et al. pointed out that 
such impairment in oxidative flexibility was mostly related to the decreased transport of 
glucose to muscle and liver cells (due to insulin resistance) rather than a true cellular 
oxidative defect [2].   
 
To understand how intramyocellular lipids accumulate and cause insulin resistance, the 
assessment of metabolic flexibility to a high-fat diet is more relevant than during a 
hyperinsulinemic clamp. Impaired capacity to upregulate muscle and liver lipid oxidation in 
face of high-fat availability will lead to increased muscle/hepatic fat accumulation and thus to 
insulin resistance. Surprisingly, very few studies have investigated the macronutrient oxidized 
in response to high-fat diets (either single meal or prolonged change in macronutrients 
composition) [3-7]. Understanding the impact of different meal compositions—specifically, 
high-fat vs. low-fat—on fuel oxidation is important and could lend itself to future dietary 
prescriptions based on physiologic responses. However, no studies have examined the 
physiological metabolic flexibility that occurs in response to a single dinner meal and the 
subsequent drop in RQ that occurs during sleep until the morning awakening.  
 
Using preliminary data that assessed individual adaptation to a high-fat diet in a 
respiratory chamber [the ADAPT study; 3], we were 
able to quantify metabolic flexibility during an overnight 
stay in 8 subjects following both a standard-fat and 
high-fat meal (see Figure 1). While both meal-
stimulated and sleep changes in RQ were observed, 
the study was not designed to adequately assess 
these changes in a very controlled manner since there 
was no instructions regarding posture and 
spontaneous physical activity. Therefore, in a more 
controlled protocol specifically designed to assess 
metabolic flexibility (measure of baseline and post-
meal energy metabolism in sitting position with the 
least possible motion), we will characterize changes 
in RQ in healthy, young men and women during an 
overnight stay in a respiratory chamber following a 
low-fat (high-CHO) or high-fat meal using a 
randomized, crossover study design. The study will establish a novel paradigm to 
investigate true physiological metabolic flexibility in response to meals of different 

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macronutrient compositions. The switch from fat to CHO oxidation in response to a 
standardized meal, as well as the rapidity at which the body starts oxidizing fat during times 
of fasting (sleep) will be examined. The proposed study will provide preliminary data for 
future studies measuring metabolic flexibility in individuals with or without obesity/insulin 
resistance, and pioneer the development of novel physiological approaches of metabolic 
flexibility assessment and its implications in the development of metabolic diseases. 
2. 
STUDY AIM: 
The overarching aim of this study: To determine the effect of different meal compositions 
(high- vs. low-fat) on metabolic flexibility as it relates to meal-stimulated (ΔRQ1) and sleep 
(ΔRQ2) metabolic flexibility, as well as the time course changes in RQ (i.e., peak RQ, time to 
peak RQ, nadir RQ, time to nadir, slope, area under the curve). 
 
Hypotheses: 
Hypothesis 1: Healthy adults will have greater changes in RQ following a low-fat (high-
CHO) meal compared to a high-fat meal. 
 
Hypothesis 2: Healthy adults will have a faster (shorter) time to peak RQ and time to 
sleep metabolic flexibility following a low-fat (high-CHO) meal compared to a high-fat 
meal. 
 
 
3. 
RESEARCH DESIGN 
This is a randomized, crossover study, 8 healthy individuals will complete two overnight stays 
in a respiratory chamber: once while eating a low-fat (high-CHO) meal (20% fat) and once 
while eating a high-fat meal (60% fat). Primary endpoints include 2 separate measures of 
metabolic flexibility—the 4-h meal-stimulated RQ and the subsequent drop in RQ during 
sleep. Secondary endpoints include time course changes in RQ.  
 
 
 
 
4. 
STUDY POPULATION 
 
4.1 
Participants 
We will enroll up to 10 healthy men or women (goal n=8 completers; 4 male, 4 female). If 
eligible and enrolled, all individuals will undergo all testing procedures.  
 
4.2 
Inclusion Criteria 
Eligibility criteria include: 
 
Healthy male or female 
Figure 2. Schedule of Metabolic Chamber Measurements 
Continuous Glucose Monitoring 

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 
Ages 18-40 y 
 
BMI between 20 kg/m2 and 30 kg/m2 (±0.5 kg/m2 will be accepted) 
 
Are willing to consume pre-prepared meals 
 
Are willing to maintain current physical activity, sleep schedule, and dietary habits 
during the study. 
 
Medically cleared for participation in the study by Medical Investigator 
 
4.3 
Exclusion Criteria 
Participants are ineligible to participate (or will be excluded from participating in this 
study) if they meet any of the following criteria: 
 
Unstable weight in the last 3 months [gain or loss >7 lb (or 3.2 kg)] 
 
Currently working shift work  
 
Smoking or use of tobacco products within the last 3 months 
 
Ammenorrhea (or absence of regular monthly cycles) 
 
History of clinically diagnosed diabetes or a fasting blood glucose >126 mg/dL 
 
Average screening blood pressure >140/90 mmHg 
 
Previous bariatric surgery (or other surgeries) for obesity or weight loss 
 
Use of medications affecting metabolism or sleep  
 
History of neurological disease 
 
History of cardiovascular disease (or other chronic diseases) 
 
Pregnant, planning to become pregnant, or breastfeeding 
 
Adherence to special restrained diets (e.g., low-CHO, low-fat, or vegetarian/vegan 
diets) over the last 3 months. 
 
5. 
STUDY DESCRIPTION 
 
5.1 
Recruitment 
Potential participants will be recruited through PBRC via IRB approved recruitment 
materials (e.g., landing page, listserv, social media). Individuals can either complete the 
webscreening form directly from the PBRC landing page, call PBRC directly, or e-mail 
the Recruitment Core. Potentially eligible participants will then undergo a phone screen 
to answer a series of yes or no questions regarding their age, gender, body weight and 
height, menopause symptom experience, presence of disease, and smoking status. 
Eligible individuals will be scheduled for a screening visit at PBRC. 
 
5.2 
Location 
All clinic visits will occur at Pennington Biomedical Research Center (PBRC). 
 
5.3 
Consent Process 
The informed consent process will be conducted at PBRC screening visits and will be 
conducted primarily by the study coordinator, but also on occasion by the study PI (Dr. 
David McDougal) or by a trained clinic staff. Written consent will be obtained before any 
procedures are performed. Potential subjects will be given ample time to read the 
Informed Consent and allowed to ask questions about the study. 
 
5.4 
Screening Process 

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Interested participants will respond to targeted recruitment materials for initial screening 
by study staff via web, phone and/or email. Following determination of initial eligibility 
(e.g., age, weight, and height), a fasting screening visit (SV1) will occur at PBRC. 
 
Participants will provide informed consent prior to the initiation of study procedures. 
Height, weight, and vital signs will be measured. Medical history will be reviewed for 
exclusionary criteria and a screening health questionnaire will be administered. In 
addition, participants will have blood drawn for measurement of general health (Chem15 
including lipid panel, and CBC). Current (or recent) medication usage will be recorded. If 
inclusion/exclusion criteria are satisfied, eligible participants will be notified by phone and 
a future study visit will be scheduled (Visit 1). 
 
6. 
ASSESSMENT SCHEDULE AND PROCEDURES 
An overview of all clinical assessments is presented in Table 1. Following confirmation of 
eligibility via a designated screening visit, three additional study visits including two overnight 
visits will be administered. Women will be scheduled for the two overnight visits during the 
follicular phase of their menstrual cycle (based on self-report of the date of their last period or 
oral contraceptive pack schedule).  
 
  
Table 1. Schedule of Clinic Assessments 
  
Screening  
Visit (SV) 
Visit 1 
5-14 DAY Interval 
Chamber 
1  
(Visit 2) 
5-7 DAY Interval‡ 
Chamber 
2 
(Visit 3) 
Visit Type 
Outpatient 
Outpatient 
Inpatient 
(Overnight) 
Inpatient 
(Overnight) 
Informed Consent 
X 
  
  
  
Randomization (post-screening) 
 
  
 X 
  
Prior/Concomitant Medications 
X 
X  
X 
X 
Height*, Weight, BMI 
X 
X 
X 
X 
Vital Signs (blood pressure, heart rate) 
X 
  
  
  
Fasting Blood Draw (glucose, lipids, CBC) 
X 
 
  
  
Screening Health Questionnaire 
X 
 
  
  
iDXA 
 
X  
  
  
Run-In Meals (36-h prior to Visit 2 and 3) 
 
 
X 
 
X 
 
12-h Respiratory Chamber 
  
 
 
X 
 
X 
Meal (i.e., low-fat, high-fat) 
  
 
X (E) 
X (E) 
Blood draws† 
 
 
X (E) 
X (E) 
Continuous glucose monitoring 
 
 
X (E) 
X (E) 
Urine Collection 
 
 
 
X (E) 
 
X (E) 
E=evening;  
*Height will only be measured during the Screening Visit 
†During both Visit 2 and 3, blood samples will be collected at -5, 30, 60, 90, 120, and 180 mins during the 
meal (i.e., 6 blood draws total). 
‡For female participants the interval between the two chamber visits will be variable to accommodate 
scheduling both visits during the follicular phase of their menstrual cycle.  
 
 

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7. 
STUDY VISITS 
The study visits are described in this section, while the study procedures that will be 
performed during these visits are detailed in the latter section “Study Procedures” (see 
Section 8). There will be 1 screening visit, 1 outpatient visit, and 2 inpatient overnight visits. 
Interested participants will be asked to abstain from alcohol consumption 24 hours before, 
and caffeine consumption 12 hours before their Screening Visit. The Screening Visit and Visit 
1 will be fasting visits, while Visits 2 and 3 will require participants to only eat pre-prepared 
meals on the day before and day of the visits and to stop all exercise 24 hours before the 
visits.  
 
Screening Visit (SV; Outpatient): Participants will undergo consenting and a fasting blood 
draw to assess glucose, lipid panel, and CBC. Vital signs (blood pressure, heart rate) will be 
measured.  
 
Visit 1 (Outpatient): Body composition (iDXA) will be measured. Participants will be 
scheduled for this visit 5-7 days prior to Visit 2.   
  
Visit 2 and 3 (Inpatient): Each participant will complete two inpatient stays in a respiratory 
chamber while consuming one meal (dinner). The two visits will only differ on the 
macronutrient composition of the dinner provided:   
• 
Low-fat (high-CHO) meal – 20% fat, 60% CHO and 20% protein 
• 
High-fat meal – 60% fat, 20% CHO and 20% protein 
 
The designated test dinner will be prepared by the metabolic kitchen and represent 40% of 
the daily energy requirement calculated on the basis of doubly-labeled water equations 
developed in a similar population [8]. Participants will be randomized to receive either the 
high-fat or low-fat meal during Visit 2.  The participants will then receive the other meal (high-
fat or low-fat) during Visit 3. A stratified randomization will be used to insure an equal number 
of males and females receive the high-fat and low-fat meals during Visits 2 and 3. 
 
Visit 2 and 3 will be separated by a 7-day interval in men and a 5 to 7-day interval in women 
to accommodate scheduling during the follicular phase of their menstrual cycle. 
 
Participants will be provided run-in meals for 36-h prior to each inpatient stay (i.e., breakfast, 
lunch, and dinner the day before testing, as well as breakfast and lunch the day of the 
testing) and to drink only water. Participants will be instructed to eat the provided lunch by 
1pm on the day of the visit. Participants will instructed to pick up their meals up to 2 days 
prior to the run-in period (4 days before the study visit). 
 
Participants will be scheduled to arrive at approximately 4:30pm the day of the visits. 
Approximately one hours prior to entering the metabolic chamber, participant’s will outfitted 
with a continuous glucose monitoring system (CGM).and an intravenous catheter will be 
placed in an antecubital vein for blood collection. The participant will enter the chamber at 

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approximately 5:30 pm and consume the meal at 
approximately 7pm. A total of six IV blood draws will be 
made (-5, 30, 60, 90, 120, and 180 mins relative to start of 
meal) to measure insulin, glucose, and free fatty acid 
levels (see Figure 2). All blood collections (either IV or 
finger stick) which occur while the participant is in the 
chamber will be accomplished by having the participant 
either extend their arm through one of the access ports on 
the side of the chamber or by having the IPU staff reach 
into the chamber through the access ports  (See Figure 
3).  A rubber gasket in the port prevents excessive loss of 
air from the chamber. 
 
8. 
STUDY PROCEDURES 
 
8.1 
Questionnaires 
Participants will complete a Screening Health Questionnaire (to assess general health) at 
Visit 1. 
 
8.2 
Blood Draw 
A fasting blood sample will be collected at the Screening Visit (SV) to assess overall 
health (Chem15 including lipid panel, and CBC). These measurement will require 
approximately 7.5 ml of blood to be drawn.  
 
8.3 
Dual-Energy X-ray Absorptiometry (DXA) 
The DXA scans will be performed at the Imaging Facility of PBRC at Visit 1. Total 
adiposity and regional fat mass will be assessed with DXA using a whole-body scanner 
(Lunar iDXA; General Electric, Milwaukee, WI). The DXA protocol requires that subjects 
lie on a table wearing a hospital gown and with no metal objects on them while both legs 
will be placed together using two Velcro straps. The scanner emitting low energy X-rays, 
and a detector passes along the body. The scan takes ~10 minutes and the radiation 
dose is less than 1 mrem, equal to about 12-h of background radiation. The scans will be 
analyzed with the latest software. The software version is enCORE 13.4. We will run 
quality control scans on a daily basis, and GE has indicated that accuracy of the data is 
confirmed with these daily QC scans. A pregnancy test (urine) will be given before DXA 
scans to confirm absence of pregnancy. 
 
8.4 
Continuous Glucose Monitoring  
Interstitial glucose will be assessed using continuous glucose monitoring (CGM). Briefly, 
the abdominal area will be disinfected, and then trained staff from the Inpatient Unit will 
insert a glucose sensor under the skin in the abdominal area. The sensor has a small 
needle-like probe that inserts into the subcutaneous fat of the abdomen and that 
measures blood glucose levels without removing blood from the body. The sensor will 
then be attached to the recording unit, and the set-up will be secured with adhesive to the 
participant’s body. After an initial period of equilibration with interstitial glucose, the sensor 
will be calibrated about every 2-12 hours by pricking the participant’s finger to measure 
capillary blood glucose. The CGM device records blood sugar levels every 5 minutes. 
Figure 3- Chamber access ports. 

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8.5 
Metabolic Chamber  
Subjects will enter the chamber at approximately 5:30 pm and exit the following morning 
at 7 am. The chamber is a room about 12’ x 14’ with 2 windows, a bed, a desk and chair, 
a treadmill, a TV/VCR/DVD, a computer with internet access, a telephone, toilet facilities, 
motion sensors and a camera. Participants will be able to contact the nurse or chamber 
personnel by intercom, pager or phone at any time. They will be served 1 meal while they 
are in the chamber. The sophisticated ventilation system allows their oxygen / carbon 
dioxide exchange to be measured, thereby showing the number of calories the participant 
is burning. Energy expenditure and oxidation of CHO, fat, and protein will be calculated 
based on previous work [9] (See Figure 4). Physical activity (% motion) will be assessed 
by infrared radar. Only minutes where activity is <1% will be used for calculating sleep 
data (2AM to 5AM). Adjustments will be made to the 4-h meal-stimulated period later if 
motion is detected. Baseline RQ will be defined as the mean RQ calculated during the 
last 30-min of the controlled, 45-min pre-meal RMR period. Peak RQ will be the highest 
mean, continuous 30-min RQ following the test meal, while Nadir RQ will be the lowest 
mean, continuous 30-min RQ during sleep. The difference between baseline RQ and 
peak RQ (i.e., ΔRQ1), between peak RQ and nadir RQ (i.e., ΔRQ2), and the time it takes 
to achieve these changes in RQ (i.e., Time to Peak, Time to Nadir, and Time to Sleep 
Metabolic Flexibility) will be calculated. While in the chamber, participants will collect all 
their urine for measurement of urinary nitrogen. Participants will be provided with 
breakfast after exiting the chamber.  
 
 
 
 
 
8.6 
Response to a Standardized Meal Test  
An indwelling catheter will be inserted into an antecubital vein to obtain venous blood 
samples. Participants will consume a test meal consisting of 20% fat, 60% CHO and 20% 
protein (low-fat meal) or 60% fat, 20% CHO and 20% protein (high-fat meal). The meal 
will be consumed while they are in the metabolic chamber under supervision in the 
Figure 4. Protocol for Overnight Stay in the Metabolic Chamber 

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inpatient unit. Blood will be collected in tubes according to the following schedule: -5, 30, 
60, 90, 120, and 180 mins relative to the start of the meal for measuring glucose, insulin, 
and free fatty acids.  These measurement will require approximately 27 ml of blood to be 
drawn.  
The estimated total blood volume required of each participant in the study is 61.5 ml. 
 
9. 
SUBJECT PAYMENT 
Participants will receive up to $425 upon completion of the study. If participants do not 
complete the entire study, they will be compensated $50 for completing Visits 1, $175 for 
completing Visits 2, and $200 for completing Visit 3. Participants will not be compensated for 
the Screening Visit. Participant payments will be received via check requested from the 
PBRC (LSU) payroll department. There is no compensation available for research-related 
injury. 
 
10. 
PROTECTION OF HUMAN SUBJECTS 
 
10.1 Risks to Participants 
This Human Subjects Research meets the definition of a Clinical Trial. This study does not 
involve major risk to participants. Efforts to minimize the potential risks of the assessment 
methods and outcome variables include frequent monitoring by the investigators to assure 
that no volunteer suffers any adverse effects from participating in the research.  
 
The known risks, inconveniences, or side effects from the proposed procedures in the 
project are shown: 
 
Potential Risks and Efforts to Minimize the Risks 
Procedure 
Potential Risks 
Efforts to Minimize the Risks 
Fasting for 10-h 
Nausea.  
Light snacks will be available to eat 
once fasting procedures completed. 
Questionnaires 
Uncomfortable answering questions. 
Participants may choose to skip any 
questions. 
Blood Draws 
(Fasting) 
Bruising, bleeding, pain, and infection 
pose minimal risks.  
Trained phlebotomists and personnel 
will use sterile technique. 
Dual-Energy X-ray 
Absorptiometry 
(DXA) scan 
DXA, using a whole-body scanner, 
measures the amount of bone, muscle, 
and fat in the body. The expected dose 
of radiation is minimal, equivalent to 
less than 0.0004mSv or ~12 hours of 
exposure to the sun. 
Exposure to X-rays is expected but 
minimal. A pregnancy test will be 
performed before the DXA scans on 
females of child-bearing potential.     
IV Procedure 
There is a possibility of pain, bruising, 
or infection at the site of the needle 
insertion for the IV line. 
Trained personnel minimize this risk.   

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Continuous 
Glucose 
Monitoring (CGM) 
 
 
 
 
Because CGM involves the placement 
of an implantable device below the 
skin, there is the possibility of 
discomfort, pain, and bruising at the 
site where the device is inserted. There 
is also a small risk of bleeding and a 
very small risk of infection at the site of 
the blood draw. Finally, the adhesive 
may cause redness or irritation of the 
skin. 
Aseptic (sterile) technique and trained 
personnel minimize these risks.   
 
Metabolic 
Chamber 
(Overnight) 
Participants may experience some 
level of claustrophobia or discomfort 
from staying in the chamber and being 
continuously monitored by a camera. 
The camera has been installed for the 
participant’s safety and no one is 
allowed access to the monitor except 
chamber personnel. 
Participants can open the door of the 
chamber in case of an emergency.  
Trained personnel also minimize this 
risk.   
 
 
 
Confidentiality of 
Data 
Taking part in this research may 
involve providing information that one 
considers confidential or private. There 
is a slight risk that data could be 
revealed inappropriately or accidentally  
Study researchers and staff will take 
steps to protect data that is collected.  
Efforts, such as coding research 
records, keeping research records 
secure and allowing only authorized 
people to access research records, 
will be made to keep the data safe. 
 
10.2 Safety Monitoring 
In this study, an adverse event or experience is defined as any health-related unfavorable 
or unintended medical occurrence that happens throughout study participation. Examples 
of adverse events include but are not limited to the following:  
 
A clinically significant laboratory or clinical test result. 
 
An event that results in missing a study visit. 
 
An event that requires a visit to a physician. 
 
An event that occurs as a result of a study procedure. 
 
Unanticipated or untoward medical events that may be study related. 
 
Pennington Biomedical Research Center is an AAHRPP accredited institution and, above 
all else, is committed to ensuring and maintaining the safety of its participants. We will use 
the provided definitions of Adverse Events and Serious Adverse Events. Events will be 
reported according to our institutional reporting policy. 
 
A serious adverse event (SAE) is defined as an unanticipated medical occurrence that is 
deemed associated with study participation by the study Medical Investigator that results in 
one of the following: 
 
Death 
 
Life-threatening event 

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o Life threatening events in participants are defined as those that in the view of 
the research staff and PI put the individual patient at imminent substantial 
risk of dying, or if continued participation in the study might have resulted in 
death.   
 
Hospitalization (initial or prolonged) 
o Hospitalization or acute outpatient evaluation (e.g., in an emergency room) 
alone is not sufficient to qualify as a serious adverse event.  
o Any medical or surgical procedure performed (e.g., surgery that is not the 
planned bilateral oophorectomy, transfusion) itself is not the adverse event; 
instead, the condition that leads to the procedure is the adverse event.   
 
Disability or permanent damage 
o If the adverse event resulted in a substantial disruption of the participant’s 
ability to conduct normal life functions, i.e., the adverse event resulted in a 
significant, persistent or permanent change, impairment, damage or 
disruption in the patient's body function/structure, physical activities and/or 
quality of life. 
 
Medical Intervention to prevent permanent impairment or damage 
 
10.2.1 Surveillance and Reporting Procedures 
All AEs from date of consent will be reported. Adverse events will be documented 
during the scheduled visits or during phone calls during off-weeks. For each sign, 
symptom or adverse event, the following information will be recorded:  
 
A brief descriptor of the adverse event  
 
Start and stop dates  
 
Intensity (mild / moderate / severe)  
 
Whether the AE was “serious” or not (as defined below)  
 
Causal association with the intervention assigned (none / doubtful / possibly / 
probably / very likely)  
 
Outcome (resolved / resolved with sequelae / improving / still present and 
unchanged / death)  
 
Action taken with respect to the intervention (none / intervention temporarily 
discontinued / medical therapy required / intervention permanently 
discontinued / other).  
 
Adverse event data will be collected from the date of consent until the final visit. 
Adverse event data will be analyzed quarterly, but serious or life-threatening adverse 
events may require immediate reporting and follow-up. We anticipate most adverse 
events will be mild and the participant will be able to resume activities within a day or 
two of reporting the event. Adverse Event reporting will follow the requirements of the 
IRB of the PBRC specified in the HRPP Policy 8.0 Unanticipated Problems Involving 
Risks to Subjects or Others. Only adverse events that qualify as unanticipated 
problems will be reported to the IRB. Unanticipated problems involving risks to 
participants or others include incidents only if the incident is unexpected, related or 
possibly related to participation in the research, and indicated that subjects or others 
are at a greater risk of harm than was previously known or recognized. Any action 

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resulting in a temporary or permanent suspension of this study (e.g., IRB actions, or 
actions by the PI and/or co-investigators) will be reported to the appropriate officials. 
 
10.3 Withdrawal of Subjects 
There is no risk associated with withdrawal from the study. Participants may be withdrawn 
from the study if either the PI or MI feels that their continued participation would jeopardize 
either the subject’s health or the results of the study. 
 
10.4 Stopping Rules 
This study does not involve major risk for participating. Nevertheless, in addition to 
monitoring recruitment, we also will monitor the rates of adverse events in our participants. 
The study investigators will alert the IRB, if a larger than reasonably expected adverse 
event rate occurs in our participants. 
 
10.5 Data Collection, Storage, and Confidentiality 
 
10.5.1 Protection of Stored Data 
Protection of subject privacy will be accomplished by a variety of stringent security 
measures. All medical records will be stored in locked areas, and access to these 
areas is limited to the clinic support staff, the Director of the clinical facilities, PIs at 
PBRC, and authorized designees of the PI. Volunteer medical records will be filed 
according to an assigned volunteer ID number. All forms on the chart, with the 
exception of the consent form, will display only the ID number, with no participant-
identifiable information. All of these will be kept separate from records with names 
and other personal information. The only people who will know that these patients 
are research participants are members of the research team. No information about 
them, or provided by them during the research, will be disclosed to others without 
their written permission, except if it is necessary to protect their rights or welfare (for 
example, in case of injury or emergency care), or if it is required by law. When the 
results of the research are published or discussed in conferences, no information will 
be included that would reveal the identity of the individuals. Participants will be 
identified by codes when the data gathered in this procedure is presented or 
published. Authorized representatives of the NIH may need to review records of 
individual participants. As a result, they may see their name; but they are bound by 
rules of confidentiality not to reveal the participants’ identity to others. Electronic data 
storage is secured with password protection and similarly restricted with only the PI 
and authorized persons having access to databases containing confidential clinical 
records (i.e. those containing name, social security number, or other identifying 
information). Electronic communication will involve only unidentifiable information.                  
 
10.5.2 Confidentiality 
PBRC complies with the federal 1996 Health Insurance Portability and Accountability 
Act (HIPAA). Specifically, PBRC protects the privacy and confidentiality of medical 
records and information contained in medical records of persons who are subjects of 
research projects, including all protected health information (PHI) as defined by the 
HIPAA privacy Regulations. PHI of research subjects and the use or disclosure of 

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such information is governed by PBRC research policies as well as Common Rule, 
FDA regulations, and other applicable laws.  
 
PBRC and study PI (the person chiefly responsible for the record) protect the privacy 
of research subjects and their PHI collected during a research project. PBRC will not 
use or disclose existing PHI or PHI created during a research project, unless the: 
 
Subject signs both (a) a HIPAA Authorization for use and disclosure of PHI 
using an approved Authorization Form or other form containing all the 
elements of legally effective HIPAA authorization; and (b) the informed 
consent to participate in research form approved by IRB; or 
 
IRB grants a waiver to the requirement of obtaining a signed HIPAA 
authorization Form, or 
 
IRB approved protocol uses properly de-identified PHI 
 
All volunteers are assured of their anonymity and confidentiality both verbally and in 
the informed consent. 
 
10.5.3 ClinicalTrials.gov Requirements 
Upon successful funding of the study, the clinical study will be registered at the 
ClinicalTrials.gov website, in accordance with NIH recommendations. The unique 
NCT identifier will be included in all future Progress Reports and publications.   
 
11. 
DATA MANAGEMENT 
 
11.1 Power Calculations 
Assuming a 10% dropout rate, this study is powered at the 83% level to detect a 
significant (α=0.05) difference between the high- and low-fat diet for the primary endpoint, 
sleep metabolic flexibility (see Fig 2). The analysis, based on data from the ADAPT study, 
assumed a sleep metabolic flexibility of 0.14 for the standard-fat meal and 0.09 for the 
high-fat meal. 
 
11.2 Statistical Analyses 
A linear mixed model accounting for both within-subject repeated measures and 
crossover effect will be used to estimate the effect of meal composition over time. Results 
will be based on t-tests using the least squared means from the mixed models. 
 
12. 
BENEFITS, RISKS, AND SAFETY 
 
12.1 
Potential Benefits 
We cannot promise any benefits from participants being in the study. However, possible 
benefits include gaining information about your overall health and body composition. The 
results of this study could also provide important information about how an individual’s 
metabolic flexibility changes following ingestion of meals with differing fat content. 
  
12.2 
Risks 
This study involves the following risks to subjects: 

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 
Fasting for 10 hours: There is a possibility that fasting for 10 hours may make 
you feel nauseous. Light snacks such as granola bars and juice are available for 
you to eat once the fasting procedures are completed. 
 
Body weight: There is no known risk associated with recording body weights. 
 
Questionnaire: Due to the sensitive nature of the questionnaire, you may skip 
any questions that you do not wish to answer. 
 
Urine collection: There are no known risks of collecting urine into a container. 
 
Blood draws: There is the possibility of infection and/or pain and bruising at the 
vein on your arm where the needle is inserted. Aseptic (sterile) technique and 
trained personnel minimize these risks.  
 
Body composition by DXA scan: The amount of radiation used for this 
procedure is very small. The radiation dose for this scan is equivalent to the 
radiation you are naturally exposed to in the environment in less than one day. 
Scans will not be performed on any subject who is pregnant. A pregnancy test will 
be performed within 72 hours before the scan on females of child-bearing 
potential.     
 
Continuous Glucose Monitoring (CGM):  Because CGM involves the placement of 
an implantable device below the skin, there is the possibility of discomfort, pain, and 
bruising at the site where the device is inserted.  There is also a small risk of bleeding 
and a very small risk of infection at the site of the blood draw.  Aseptic (sterile) 
technique and trained personnel minimize these risks.  Finally, the adhesive may 
cause redness or irritation of the skin. 
 
IV Procedure: There is a possibility of pain, bruising, or infection at the site of the 
needle insertion for the IV line. Trained personnel minimize this risk.   
 
Metabolic Chamber (Overnight):  Participants may experience some level of 
claustrophobia or discomfort from staying in the chamber and being continuously 
monitored by a camera. The camera has been installed for the participant’s safety 
and no one is allowed access to the monitor except chamber personnel. 
Participants can open the door of the chamber in case of an emergency.  
Trained personnel also minimize this risk.   
 
In addition to the risks listed above, participants may experience a previously 
unknown risk or side effect. 
 
13. 
REFERENCES 
 
 
1. 
Kelley, D.E. and L.J. Mandarino, Fuel selection in human skeletal muscle in insulin 
resistance: a reexamination. Diabetes, 2000. 49(5): p. 677-83. 
2. 
Galgani, J.E., et al., Metabolic flexibility in response to glucose is not impaired in people 
with type 2 diabetes after controlling for glucose disposal rate. Diabetes, 2008. 57(4): p. 
841-5. 
3. 
Galgani, J.E., et al., Effect of a 3-day high-fat feeding period on carbohydrate balance and 
ad libitum energy intake in humans. International journal of obesity, 2010. 34(5): p. 886-
91. 

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4. 
Schrauwen, P., W.D. van Marken Lichtenbelt, and K.R. Westerterp, Fat and carbohydrate 
balances during adaptation to a high-fat diet. The American Journal of Clinical Nutrition, 
2000. 72(5): p. 1239-41. 
5. 
Schrauwen, P., et al., Changes in fat oxidation in response to a high-fat diet. The 
American Journal of Clinical Nutrition, 1997. 66(2): p. 276-82. 
6. 
Schutz, Y., J.P. Flatt, and E. Jequier, Failure of dietary fat intake to promote fat oxidation: 
a factor favoring the development of obesity. The American Journal of Clinical Nutrition, 
1989. 50(2): p. 307-14. 
7. 
Heilbronn, L.K., et al., Impaired fat oxidation after a single high-fat meal in insulin-sensitive 
nondiabetic individuals with a family history of type 2 diabetes. Diabetes, 2007. 56(8): p. 
2046-53. 
8. 
Redman, L.M., et al., Metabolic and behavioral compensations in response to caloric 
restriction: implications for the maintenance of weight loss. PLoS ONE, 2009. 4(2): p. 
e4377. 
9. 
Jequier, E., K. Acheson, and Y. Schutz, Assessment of energy expenditure and fuel 
utilization in man. Annual review of nutrition, 1987. 7: p. 187-208.