Clinical trial NCT04766021
Conditions: Resting Energy Expenditure Thermogenesis Brown Adipose Tissue
Sponsor: Indiana University
Start date: 2021-11-30; Completion date: 2023-04-27
Enrollment: 30 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
Many Navy diving operations are performed in cold water. Despite technical advances to improve thermal protection for cold water diving, these applications are cumbersome and do not provide complete thermal protection as thermal discomfort is subjectively reported by many Navy divers. Brown adipose tissue is highly thermogenic in humans. Therefore, activation of brown adipose tissue might improve cold water tolerance and lower thermal discomfort during cold water diving operations. Mirabegron is a beta-3-adrenergic receptor agonist that is used to treat overactive bladder. Beta-3-adrenergic receptors are located on the urinary bladder, gallbladder and brown adipose tissue. Recent evidence has demonstrated that acute mirabegron administration increases thermogenesis for \~3 hours in humans. However, it is currently not known which dose of mirabegron can increase thermogenesis for longer durations. It is also not known if mirabegron administration can improve cold water tolerance and thermal discomfort during cold water immersion. Finally, it is not known if mirabegron can increase thermogenesis during sympathetic stimulation. This project will fill these knowledge gaps by determining which dose of mirabegron administration will increase thermogenesis during 6 hours of a mild cold stress challenge. This study is part of a collection of studies that will show if mirabegron is a potential ergogenic aid that can be used to improve cold water tolerance in Navy divers which will ultimately improve the likelihood of successful missions.

Detailed description:
This is a randomized, double-blind, placebo-controlled, cross-over experimental design. Participants will be asked to complete 5 visits to the laboratory; one informed consent/screening visit and 4 study visits. On study visit days, participants will report to the laboratory following a 12-hour fast, and 24-hour abstention from exercise, caffeine, and alcohol. During the study visits, participants will be instrumented for skin temperature (12-sites: calf, shin, front of the thigh, back of the thigh, chest, upper back, forehead, lower back, abdomen, forearm, hand, and foot; iButtons) core temperature (rectal thermistor), indices of shivering (surface mechanomyography using triaxial accelerometers at 3 anatomic sites (chest, upper back, and thigh) and the bedside shivering assessment scale), brachial artery blood pressure (brachial artery auscultation), and heart rate (3-lead ECG). Thermal perceptions will be assessed using Likert scales for thermal discomfort and thermal sensation. Participants will be shirtless (sports bra for women) and will wear shorts throughout the study. After 20 minutes of quiet resting in the supine position, 5 minutes of baseline measurements will be taken, thermal perceptions and an infrared thermography image of the supraclavicular fossa will be obtained as an indicator of brown adipose tissue activation (6), and a whole blood sample will be obtained.

Participants will then ingest mirabegron (100 mg, 150 mg, or 200 mg) or placebo. Thirty minutes after mirabegron or placebo has been ingested, participants will enter the whole-body indirect calorimeter; the internal temperature of the calorimeter will be set to 20 degrees C (68 degrees F). This will elicit a mild cold stress over the 6 hours of observation in the whole-body indirect calorimeter. The whole-body indirect calorimeter provides an accurate and continuous measure of thermogenesis (in the form of energy expenditure) that can be used over long periods of time in a stable environment. The whole-body calorimeter is designed to perform human studies that are up to 48 hours in duration and contains a private washroom. While in the whole-body indirect calorimeter, participants will be instructed to be sedentary and will be allowed to watch television or read. In order to stay consistent between study visits, participants will be instructed to spend the same amount of time watching television or reading for each study visit. Every 30 minutes, we will record skin temperatures, core temperature, blood pressure, and heart rate. Thermogenesis will be determined by having participants lie supine for 30 minutes and resting energy expenditure will be calculated using the abbreviated Weir formula every hour (5). Thermal perceptions and an infrared thermography image will also be obtained at these time points. At approximately the midpoint of the 6-hour measurement period, participants will have the opportunity to use the restroom and an additional urine sample will be collected at this time. At the end of the 6-hour measurement period, participants will exit the whole-body indirect calorimeter and final measurements of skin temperature, core temperature, heart rate, and blood pressure will be obtained as well as a final infrared thermography image and a whole blood sample. After the experiment is complete, participants will be given a mylar blanket to re-warm themselves prior to leaving the laboratory.

Participants will be asked to return to the laboratory after 10-14 days to repeat the experiment until all 4 dosing conditions (placebo, 100 mg, 150 mg, and 200 mg of mirabegron) have been tested. Each participant's order of assignment to placebo or drug dosing over the 4 study visits will be randomly assigned.

Arms: arm 1: Given once, followed by observation for 6 hours in a 20 degree Celsius (68 degree Fahrenheit) room arm 2: Given once, followed by observation for 6 hours in a 20 degree Celsius (68 degree Fahrenheit) room arm 3: Given once, followed by observation for 6 hours in a 20 degree Celsius (68 degree Fahrenheit) room arm 4: Given once, followed by observation for 6 hours in a 20 degree Celsius (68 degree Fahrenheit) room
Interventions: intervention 1: Mirabegron intervention 2: Placebo
Intervention descriptions: intervention 1: Dose-response effect on thermogenesis intervention 2: Placebo control condition

Protocol documents:
Prot_SAP_000.pdf: 
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Mirabegron and Physiological Function in Cold Environments 
 
NCT04766021 
 
January 29, 2024 
 


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Mirabegron and Physiological Function in Cold 
Environments 
 
Blair Johnson, PhD, Associate Professor 
PH 042, Department of Kinesiology, School of Public Health-Bloomington 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Co-Investigator: 
Dr. Andrew Watters, MD, IU Emergency Health Services 
 
 
Support Provided by: 
Office of Naval Research 
 
 
IND/IDE#: 
(IND Exemption PIND 153764) 
 
 
 


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Table of Contents: 
 
Study Schema 
1.0 
Background & Rationale 
2.0 
Objective(s) 
2.1 
Primary Objective 
2.2 
Secondary Objective 
2.3 
Tertiary/Exploratory/Correlative Objectives 
3.0 
Outcome Measures 
3.1 
Primary Outcome Measures 
3.2 
Secondary Outcome Measures 
3.3 
Tertiary/ Exploratory/ Correlative Outcome Measures 
4.0 
Eligibility Criteria 
4.1 
Inclusion Criteria 
4.2 
Exclusion Criteria 
5.0 
Study Design 
6.0 
Enrollment/Randomization 
7.0 
Study Procedures 
8.0 
Study Calendar 
9.0 
Reportable Events 
10.0 Data Safety Monitoring 
11.0 Study Withdrawal/Discontinuation 
12.0 Statistical Considerations 
13.0 Data Management 
14.0 Privacy/Confidentiality Issues 
15.0 Follow-up and Record Retention 
16.0 References 
17.0 Appendix 
 


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Abbreviations 
This page is optional. The list below includes some common abbreviations. However, this list 
should be customized for each protocol (i.e., abbreviations not used should be removed and 
new abbreviations used should be added to this list). 
 
ECG 
Electrocardiogram 
DEXA 
Dual-energy X-ray absorptiometry 
IPAQ 
International Physical Activity Questionnaire 
PAR-Q+ Physical Activity Readiness Questionnaire Plus 
 
 
 


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1.0 
Background & Rationale 
 
Specialized Navy personnel are required to undertake duties while immersed in cold water for 
prolong periods. These dives regularly require the use of external thermal protection (i.e., wet 
suits and dry suits). However, external thermal protection during prolonged cold-water diving is 
often times insufficient to maintain body temperature and thermal comfort, thereby potentially 
negatively impacting the safety and success of diving missions. Accordingly, developing an 
alternative strategy that can improve tolerance to cold-water immersion is of important interest 
to Navy divers and special forces. 
 
Mirabegron (Myrbetriq®, extended-release tablet, Astellas Pharma), is a medication approved by 
the Food and Drug Administration for the treatment of overactive bladder; the mechanism of 
action is stimulation of the beta-3-adrenergic receptor (1). However, mirabegron has additional 
effects that include the activation of beta-3-adrenergic receptors located on brown adipose 
tissue (1). Brown adipose tissue is considered to be highly thermogenic and contributes to non-
shivering thermogenesis (i.e. the generation of heat in the absence of movement). Recent 
evidence indicates that resting energy expenditure (a direct reflection of thermogenesis) and 
supraclavicular skin temperature (an indicator of brown adipose tissue activation) are elevated 
for 3-4 hours after the acute administration of mirabegron in single doses ranging from 100 mg 
to 200 mg (2). However, it is not known which dose of mirabegron exerts the longest lasting 
thermogenic effects. Importantly, plasma concentrations of mirabegron peak 3-4 hours after 
administration (3, 4) and the biological half-life of mirabegron is 40-50 hours (4). Thus, the 
thermogenic effects of mirabegron might extend past 4 hours. This is important because many 
cold-water dives last longer than 4 hours. 
 
2.0 
Objective(s) 
2.1 
Primary Objectives 
Specific Aim 1: 
a. Determine if acute mirabegron administration will increase thermogenesis during 6 
hours of a mild cold stress challenge. 
b. Determine the dose of acute mirabegron administration that will produce the greatest 
increases in thermogenesis during 6 hours of a mild cold stress challenge. 
 Specific Aim 2: 
a.  Determine if acute mirabegron administration will delay the fall in core temperature 
during a progressive cold-water immersion challenge. 
b. To determine if acute mirabegron administration will delay the onset of shivering during 
a progressive cold-water immersion challenge. 
 
2.2 
Secondary Objective 
Specific Aim 1: 
Determine if core temperature, mean skin temperature (6 thermocouples), heart rate 
(ECG), blood pressure (brachial artery auscultation), indices of shivering (e.g., oxygen 
consumption, surface mechanomyography (using 3 triaxial accelerometers) and the 
bedside shivering assessment scale), thermal perceptions (thermal comfort and thermal 
sensation), and brown adipose tissue activation using infrared thermography are 
influenced by the dose of mirabegron 6 hours of a mild cold stress challenge. 
 
 


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 Specific Aim 2: 
Determine if the shivering inflection point during the progressive cold-water immersion 
challenge is shifted following mirabegron ingestion.  
 
2.3 
Tertiary/Exploratory/Correlative Objectives 
Specific Aim 3: 
To determine if variations in gut microbiome and their metabolites between participants 
explains the variability associated with different doses of acute mirabegron 
administration. 
 
We will correlate measures of body composition derived from dual-energy X-ray 
absorptiometry (DEXA) to primary and secondary outcome measures. 
 
3.0 
Outcome Measures/Endpoints 
3.1 
Primary Outcome Measures 
Specific Aim 1: 
Thermogenesis (i.e., resting energy expenditure) and thermogenesis area under the curve 
during the protocol. Thermogenesis will be measured using whole body indirect 
calorimetry, calculated using the Weir formula (5), will be measured every 30 minutes 
during the 6 hour protocols. Area under the curve for thermogenesis will be calculated to 
reflect the cumulative effect of mirabegron over time. 
Specific Aim 2: 
Core temperature deflection point during the progressive cold-water immersion challenge. 
Core temperature will be continuously measured using rectal thermistors. The point at 
which core temperature begins to fall (i.e., deflection point) will be identified using Prism 
8 software by plotting core temperature vs. water temperature. 
 
Specific Aim 3: 
Gut microbiota phylotypes, fecal mirabegron concentration, and serum mirabegron 
concentration will be measured from samples obtained during the study (serum 
samples: pre-mirabegron/placebo and following 6 h of mild cold exposure; fecal 
samples: first bowel movement following 6 h of mild cold exposure). Samples will be 
assessed using bacterial DNA extraction and purification, polymerase chain reaction, and 
MinION analysis as well as metabolomics.  
 
3.2 
Secondary Outcome Measures 
Specific Aim 1: 
Core temperature, mean skin temperature (6 thermocouples), heart rate (ECG), blood 
pressure (brachial artery auscultation), indices of shivering (e.g., oxygen consumption, 
surface mechanomyography (using 3 triaxial accelerometers) and the bedside shivering 
assessment scale), thermal perceptions (thermal comfort and thermal sensation), and 
brown adipose tissue activation using infrared thermography  
Specific Aim 2: 
The shivering inflection point during the progressive cold-water immersion challenge. 
Shivering will be assessed via oxygen consumption, surface mechanomyography (using 3 
triaxial accelerometers), and the bedside shivering assessment scale. The core 
temperature immediately prior to the onset of shivering during the progressive cold-water 
challenge will be identified using Prism 8 software by plotting shivering vs. core 
temperature. 


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3.3 
Tertiary/Exploratory/Correlative Outcome Measures 
Specific Aim 3: 
Serum mirabegron metabolites will be used to determine the mirabegron metabolism 
using metabolomics.  
 
Body composition will be measured using DEXA once after informed consent during the 
screening visit. We will run exploratory correlational analyses among the other 
physiological data collected and body composition. 
 
 
4.0 
Eligibility Criteria 
4.1 
Inclusion Criteria 
List the criteria:  
• 
Men and women 
• 
18-40 years old 
• Participate in 150 minutes or more of at least moderate intensity exercise 
per week during the previous 2 years 
 
4.2 
Exclusion Criteria 
List the criteria:  
• Any autonomic cardiovascular, metabolic, neurologic, endocrine, or respiratory 
disease 
• Previously diagnosed liver and/or kidney dysfunction 
• Women who are pregnant or breastfeeding  
• Individuals currently taking a medication that cannot be safely discontinued for 
5 biological half-lifes prior to each study visit based on consultation with the 
study physician.  Birth control (including hormonal contraception) will be 
permitted despite potential to influence vasomotor activity. 
-Increases in heart rate and blood pressure are common with the higher 
doses of mirabegron that we will be testing. Additionally, medications that 
influence vasomotor activity may also influence thermoregulation. 
Therefore, for participant safety, we do not want to study participants that 
are taking a medication that might interfere with our ability to properly 
monitor and interpret the potential side effects of mirabegron during cold 
exposure. 
• Current tobacco or electronic cigarette use or consistent use within the last 1 
year 
 
5.0 
Study Design 
 
Specific Aim 1: This is a randomized, double-blind, placebo-controlled, cross-over experimental 
design. Participants will be asked to complete 5 visits to the laboratory; one informed 
consent/screening visit and 4 study visits. During the informed consent/screening visit, 
participants will complete the informed consent, eligibility criteria will be presented, resting 
blood pressure and heart rate (derived from the blood pressure measurement) will be 
measured, and participants will complete the following questionnaires: iPAQ, PAR-Q, and Health 


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History Questionnaire. During the study visits, participants will undergo baseline measurements 
(described in detail in Section 7.0) followed by ingesting either a placebo or 3 different doses of 
mirabegron (100 mg, 150 mg, or 200 mg). Mirabegron will be prescribed by our study 
physician, Dr. Andrew Watters. Dr. Watters is a physician in the Department of Emergency 
Medicine at Indiana University Health and he is a Fellow in Wilderness Medicine through the 
Wilderness Medical Society as well as a member of the Indiana Task Force 1 (part of the FEMA 
Urban Search and Rescue System. Dr. Watters will determine if it is safe for a potential 
participant to stop their medication for 5 half-lifes during screening. Dr. Watters will also review 
health history, resting heart rate and blood pressure measurements, and contraindications for 
mirabegron use to determine eligibility. A second set of baseline measurements will be made 
prior to participants entering our whole-body indirect calorimeter set at 20C (68F) for 6 hours. 
Every 30 minutes in the chamber, participants will be asked to assume the supine position to 
record resting energy expenditure (i.e., thermogenesis) and other physiological variables 
(described in detail in Section 7.0). This design will allow us to determine which dose of 
mirabegron produces the greatest increase in thermogenesis. 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
Specific Aim 2: This is a randomized, double-blind, placebo-controlled, cross-over experimental 
design. Participants will be asked to complete 3 visits to the laboratory; one informed 
consent/screening visit and 2 study visits. During the informed consent/screening visit, 
participants will complete the informed consent, eligibility criteria will be presented, resting 
blood pressure and heart rate (derived from the blood pressure measurement) will be 
measured, and participants will complete the following questionnaires: iPAQ, PAR-Q, and Health 
History Questionnaire. During the study visits, participants will undergo baseline measurements 
(described in detail in Section 7.0) followed by ingesting either a placebo or one dose of 
mirabegron (100 mg) prior to entering our water immersion tank. Mirabegron will be prescribed 
by our study physician, Dr. Andrew Watters (Department of Emergency Medicine, Indiana 
University Health). The dose of mirabegron that will be used for Specific Aim 2 is dependent on 
our findings from Specific Aim 1. The dose of mirabegron that elicits the greatest increase in 
thermogenesis in Specific Aim 1 will be used for Specific Aim 2. A second set of baseline 
measurements will be made and then the water immersion tank will then be rapidly filled with 
35C (95F) water. The temperature of the water will be progressively lowered by ~12C every 
60 minutes until the water temperature reaches 10C (50F) or until the participant can no 
longer tolerate the cold or if rectal temperature reaches 35.5C (95.9F). Participants will be 
continuously monitored for rectal temperature, indices of shivering, skin temperature, heart 
Informed Consent/Screening Visit:  Obtain informed consent. Screen potential 
participants by inclusion and exclusion criteria.  Obtain Health History Questionnaire, PAR-
Q+, and IPAQ and resting heart rate and blood pressure.  Perform body composition 
assessment with DEXA.  
Study Visits 1-4 
The order of placebo and mirabegron doses (100 mg, 150 mg, and 200 mg) will be 
randomized among Study Visits 1-4. All study procedures will be identical between Study 
Visits. 
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rate, blood pressure, and thermal perceptions (described in detail in Section 7.0). This design 
will allow us to determine if mirabegron improves cold water immersion tolerance. 
  
  
 
 
 
 
 
 
 
 
 
 
 
Specific Aim 3: This aim will be addressed using the study design and visits used for Specific 
Aim 1 (a randomized, double-blind, placebo-controlled, cross-over experimental design). During 
these study visits, we will collect whole blood at pre-mirabegron/placebo and 15 minutes post 
the 6 h protocol. The whole blood will be allowed to clot and we will obtain serum samples for 
analyses. Participants will also be given fecal sample collection kits that they will take home to 
be used to collect fecal samples from their first bowel movement following each study visit. The 
serum and fecal samples will be sent to a colleague at Rutgers University for analyses. All 
samples will be de-identified prior to being sent to Rutgers University. 
 
6.0 
Enrollment/Randomization 
 
We plan to recruit 60 total participants for this project (30 for Specific Aim 1 and 30 for Specific 
Aim 2). Participants who partake in Specific Aim 1 will also be used for Specific Aim 3 and can 
participate in Specific Aim 2. Two informed consent forms will be used for Specific Aim 1 & 3 
(Mirabegron and Thermogenesis in Cold Air) and Specific Aim 2 (Mirabegron and 
Thermogenesis in Cold Water). 
 
Participants will be recruited from the IU student body and local citizen population through 
flyers placed around campus, announcements made to classes of students, and word of mouth. 
Participants who are interested will contact the principal investigator or IRB approved personnel 
by email or by phone. This population may include IU students and individuals outside the IU 
community.  
 
Potential participants will be given the basic information about the study and schedule an 
appointment for screening if they indicate that they would be interested in participating. 
Participants will be provided with detailed description of study procedures prior to study 
enrollment. Participants will be asked to read the consent form at this time and encouraged to 
ask any questions. Participants can then: 1) sign the consent form; 2) take it home for further 
consideration; or 3) decide not to participate. Participants will be screened once written informed 
consent has been given. 
 
Both Specific Aim 1 (& 3) and Specific Aim 2 study visits will be completed in a randomized order. 
Randomization of the order of the study visits will be completed by a research technician using a 
Informed Consent/Screening Visit:  Obtain informed consent. Screen potential 
participants by inclusion and exclusion criteria.  Obtain Health History Questionnaire, PAR-
Q+, and IPAQ and resting heart rate and blood pressure. Perform body composition 
assessment with DEXA.  
Study Visits 1-2 
The order of placebo and mirabegron (100 mg) will be randomized between Study Visits 1-
2. All study procedures will be identical between Study Visits. 
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randomization list that will be created by a trained statistician external to the research team using 
R (R Foundation for Statistical Computing, Vienna, Austria) and our subjects will be randomized 
by our research technician. 
 
7.0 
Study Procedures 
 
All of the below mentioned procedures are being conducted for purposes of the research.  
 
• 
Height: Body height will be measured using a stadiometer.  
 
• 
Weight: Weight will be measured using a scale in a private room.  
 
• 
Pregnancy test: To ensure female participants are not pregnant they will undertake a urine 
pregnancy test during screening and before each study visit. 
 
• 
Urine Specific Gravity: A sample of urine will be used to assess urine specific gravity using a 
refractometer. 
 
• 
Heart rate: Heart rate will be obtained using a 3-lead electrocardiogram. 
 
• 
Arm Blood Pressure: Blood pressure will be monitored using a cuff placed on the upper arm 
that is inflated and deflated periodically.  Blood pressure measurements will be taken at 
several different time points. Heart rate will also be derived from this measure during the 
informed consent/screening visit. Each measurement will last approximately 30 seconds. 
 
• 
Arterial Oxygen Saturation: Pulse oximetry will be used at the forehead to obtain continuous 
arterial oxygen saturation values. 
 
• 
Partial Pressure of End Tidal CO2: A nafion tube will be connected to a small port in the 
mouthpiece to measure end tidal partial pressure of CO2.  
 
• 
Ventilation: A pneumotach connected to a mouthpiece or facemask will be used to measure 
the flow of inspired and expired air. Expired gases will be sampled using oxygen and carbon 
dioxide sensors to determine oxygen consumption and carbon dioxide production.  
 
 
• 
Core temperature: Participants will self-insert a single use rectal temperature probe in a 
private room. A small (~3 mm in diameter), single use, lubricated, flexible probe will be 
inserted by the participant to a pre-marked depth of ~10 cm. 
 
• 
Skin Temperature: Fourteen iButtons will be adhered to the skin using adhesive tape on the 
calf, shin, front thigh, back thigh, foot, chest, upper back, lower back, forearm, hand, 
forehead, fingertip, toetip, and abdomen area to measure skin temperature. 
 
• 
Perceptual Scales: Participants will complete Likert scales to assess their thermal comfort (1 
= comfortable, 4 = very uncomfortable) and thermal sensation (1 = cold, 4 = neutral, 7 = 
hot).   
 


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• 
Bedside Shivering Scale: Two investigators will subjectively assess shivering by the 
participants using a Likert scale (0 = none, 3 = Severe) and the scores will be averaged. 
 
• 
Surface Mechanomyography: Three triaxial accelerometers adhered to the skin using 
adhesive tape on the chest, upper back, and thigh will be used to assess shivering.  
 
• 
Resting Energy Expenditure (thermogenesis): Expired oxygen, carbon dioxide, and 
ventilation will be collected from the whole-body calorimetry and used to calculate 
thermogenesis using the abbreviated Weir formula (5).  
 
• 
International Physical Activity Questionnaire: Participants will complete this form to self-
report and quantify their level of physical activity.   
 
• 
Health history and demographics questionnaire: Participants will complete a health history 
questionnaire to ensure they meet the inclusion and exclusion criteria. 
 
• 
Health history update questionnaire: Upon each visit to the laboratory participants will 
complete the heath history update questionnaire to ensure no changes in their health 
history has occurred. 
 
• 
Infrared Thermography Images: An infrared thermography camera will be used to measure 
skin temperature in the supraclavicular (i.e., collar bone) region. This provides a non-
invasive estimate of brown adipose tissue activation (6). 
 
• 
PAR-Q+: Participants will complete this questionnaire to ensure they meet the inclusion and 
exclusion criteria. 
 
• 
3-Day Dietary Log: Myfitnesspal.com (Under Armour) is a dietary log that will be used to 
quantify the type and amount of food and fluid that participants consume 3 days prior to 
each study visit. 
 
• 
Body Composition: Participants will have their body composition measured using DEXA. 
 
• 
Whole Blood Collection: 15 mL of whole blood will be obtained from an antecubital vein at 
pre-mirabegron/placebo and at 15 minutes post protocol using standard aseptic techniques 
defined by the World Health Organization. 
 
• 
Fecal Sample Collection: Participants will be given fecal collection kits (DNA Genotek) and 
will be given verbal instructions from the collection kit directions on how to obtain samples 
as well as the pamphlet that contains these directions the after the study visits of Specific 
Aim 1. Participants will be instructed to obtain samples from their first bowel movement 
after each study visit. 
 
 
 
 
 
 


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Timeline of Procedures 
 
Specific Aim 1 
• 
Informed Consent/Screening Visit: Once participants arrive at the laboratory, an 
investigator will obtain written informed consent after all procedures and study risks are 
fully explained and all questions answered. After obtaining written informed consent, 
participants will complete a Health History and Demographics Questionnaire, an 
International Physical Activity Questionnaire (IPAQ), Physical Activity Readiness 
Questionnaire (PAR-Q+). An experienced investigator will administer these 
questionnaires. These individuals can fully explain any technical terms and answer any 
inquiries related to the questionnaire. A resting heart rate and arm blood pressure 
measurement will be done after the questionnaires are complete. Participants will then 
have their body composition measured using DEXA. If DEXA cannot be performed during 
this visit, an additional visit can be scheduled. Participants will also visit with Dr. Watters 
to review medical history and resting blood pressure and heart rate measurements to 
review any contraindications for taking mirabegron. The visit with Dr. Watters can be in 
person or through a scheduled Zoom call to reduce participant scheduling burden. In the 
case of a Zoom call, Dr. Watters will be provided the medical history and resting blood 
pressure and heart rate measurements prior to the call.  
 
• 
Study Visits: Mirabegron has a half-life of ~50 hours so all study visits will be separated 
by at least a 10-day washout period. Our goal will be to have 10-14 days in between 
study visits. Participants will report to the temperature and humidity-controlled 
laboratory in the morning following a 12-hour fast, and 24-hour abstention from 
exercise, caffeine, and alcohol. Participants will bring their dietary logs for review to 
ensure a consistent diet prior to each study visit. Participants will consume a light 
standard breakfast (237 ml Ensure Original; 220 kcal) upon arrival and be instrumented 
for skin temperature (12-sites: calf, shin, front of the thigh, back of the thigh, chest, 
upper back, forehead, lower back, abdomen, forearm, hand, and foot; iButtons) core 
temperature (rectal thermistor), indices of shivering (surface mechanomyography using 
triaxial accelerometers at 3 anatomic sites (chest, upper back, and thigh) and the 
bedside shivering assessment scale), brachial artery blood pressure (brachial artery 
auscultation), and heart rate (3-lead ECG). Thermal perceptions will be assessed using 
Likert scales for thermal discomfort and thermal sensation. Participants will be shirtless 
(sports bra for women) and will wear shorts throughout the study. After 20 minutes of 
quiet resting in the supine position, 5 minutes of baseline measurements will be taken, 
thermal perceptions and an infrared thermography image of the supraclavicular fossa 
will be obtained as an indicator of brown adipose tissue activation (6), and a whole 
blood sample will be obtained. Whole blood samples will be allowed to clot at room 
temperature (~15-30 minutes). The clot will be removed via centrifuging the samples at 
1,500 g for 10 minutes. The serum samples will then be frozen (-80°C) until they are 
shipped on dry ice to Rutgers University for analysis. Participants will then ingest 
mirabegron (100 mg, 150 mg, or 200 mg) or placebo. Thirty minutes after mirabegron 
or placebo has been ingested, participants will enter the whole-body indirect 
calorimeter; the internal temperature of the calorimeter will be set to 20C (68F). This 
will elicit a mild cold stress over the 6 hours of observation in the whole-body indirect 
calorimeter. The whole-body indirect calorimeter provides an accurate and continuous 
measure of thermogenesis (in the form of energy expenditure) that can be used over 


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long periods of time in a stable environment. Our whole-body calorimeter is designed to 
perform human studies that are up to 48 hours in duration and contains a private 
washroom. While in the whole-body indirect calorimeter, participants will be instructed 
to be sedentary and will be allowed to watch television or read. In order to stay 
consistent between study visits, participants will be instructed to spend the same 
amount of time watching television or reading for each study visit. Every 30 minutes, we 
will record skin temperatures, core temperature, blood pressure, and heart rate.  
Thermogenesis will be determined by having participants lie supine for 30 minutes and 
resting energy expenditure will be calculated using the abbreviated Weir formula every 
hour (5). Thermal perceptions and an infrared thermography image will also be obtained 
at these time points. At approximately the midpoint of the 6-hour measurement period, 
participants will have the opportunity to use the restroom and an additional urine 
sample will be collected at this time. At the end of the 6-hour measurement period, 
participants will exit the whole-body indirect calorimeter and final measurements of skin 
temperature, core temperature, heart rate, and blood pressure will be obtained as well 
as a final infrared thermography image and a whole blood sample. After the study, 
participants will be given a mylar blanket to re-warm themselves prior to leaving the 
laboratory. Participants will be given a fecal collection kit that they will take with them. 
Instructions will be provided (both verbal and written) on the proper handling of their 
sample. If participants complete the informed consent/screening visit and all study visits, 
we expect that they will spend a total of ~33 hours in the study. 
 
Specific Aim 2 
• 
Informed Consent/Screening Visit: Once participants arrive at the laboratory, an 
investigator will obtain written informed consent after all procedures and study risks are 
fully explained and all questions answered. After obtaining written informed consent, 
participants will complete a Health History and Demographics Questionnaire, an 
International Physical Activity Questionnaire (IPAQ), Physical Activity Readiness 
Questionnaire (PAR-Q+). An experienced investigator will administer these 
questionnaires. These individuals can fully explain any technical terms and answer any 
inquiries related to the questionnaire. A resting heart rate and arm blood pressure 
measurement will be done after the questionnaires are complete. Participants will then 
have their body composition measured using DEXA. If DEXA cannot be performed during 
this visit, an additional visit can be scheduled. Participants will also visit with Dr. Watters 
to review medical history and resting blood pressure and heart rate measurements to 
review any contraindications for taking mirabegron. The visit with Dr. Watters can be in 
person or through a scheduled Zoom call to reduce participant scheduling burden. In the 
case of a Zoom call, Dr. Watters will be provided the medical history and resting blood 
pressure and heart rate measurements prior to the call. 
 
• 
Study Visits: Mirabegron has a half-life of ~50 hours so all study visits will be separated 
by at least a 10-day washout period. Our goal will be to have 10-14 days in between 
study visits. Participants will report to the temperature and humidity-controlled 
laboratory in the morning following a 12-hour fast, and 24-hour abstention from 
exercise, caffeine, and alcohol. Participants will bring their dietary logs for review to 
ensure a consistent diet prior to each study visit. On arrival, participants will provide a 
urine sample to ensure they are hydrated via urine specific gravity. Participants will 
consume a light standard breakfast (237 ml Ensure Original; 220 kcal) and enter the 


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empty water immersion tank. Then they will be instrumented for the measurement of 
heart rate (3-lead ECG), blood pressure (brachial artery auscultation), core temperature 
(rectal thermistor), and indices of shivering (surface mechanomyography using triaxial 
accelerometers at 3 anatomic sites (chest, upper back, and thigh), oxygen consumption 
via ventilation and expired gases, and the bedside shivering assessment scale). Thermal 
perceptions will be assessed using Likert scales for thermal discomfort and thermal 
sensation. Following 10 minutes of resting baseline measurements, infrared 
thermography will then be used to measure skin temperature of the supraclavicular 
fossa as an estimate of brown adipose tissue activation and thermal perceptions will be 
assessed. The participants will then ingest either mirabegron (100 mg) or a placebo 
tablet. The dose of mirabegron will be determined from the results of Specific Aim 1. 
The dose of mirabegron the elicits the greatest increase in thermogenesis in Specific Aim 
1 will be used in Specific Aim 2. The measurement of supraclavicular fossa skin 
temperature (infrared thermography) and thermal perceptions will be assessed every 10 
minutes prior to the progressive cold-water challenge. After 30 minutes of seated rest, 
pre-immersion measurements of heart rate, blood pressure, core temperature, 
shivering, and thermal perceptions will be conducted and an infrared thermography 
image will be taken. Then, the water immersion tank will be filled up to the participant’s 
neck with 35C (95F) water. Heart rate, blood pressure, core temperature, and 
shivering indices will be continuously measured. Infrared thermography and thermal 
perceptions will be obtained every 5 minutes throughout the progressive cold-water 
challenge. The water will be cooled at a rate of 12C per hour until the water 
temperature reaches 10C (50F), the participant can no longer tolerate the cold water, 
or core temperature reaches 35.5C (95.9F). This is 0.5C higher than the clinical 
definition of hypothermia. Also, the risks associated with hypothermia are core body 
temperature-dependent and not dependent on time (7). Upon completion of the 
progressive cold water challenge, the water in the tank will be rapidly emptied. 
Participants will gently towel dry and will be able to don a circulating warm water 
perfused suit and/or use a mylar blanket to reestablish core temperature. If participants 
complete the informed consent/screening visit and all study visits, we expect that they 
will spend a total of ~11 hours in the study. 
 
Specific Aim 3 
The serum and fecal samples that were obtained during the study visits for Specific Aim 
1 will be used for the Specific Aim 3. Serum samples will be frozen at -80C until they 
are shipped on dry ice to Rutgers University for analyses. Participants will be given the 
option to directly send their fecal samples to Rutgers University (shipping will be paid for 
by the study) or they can bring their samples in to the lab and a staff member will ship 
the samples to Rutgers University. All samples will be de-identified.  
 
8.0 
Study Calendar 
 
Specific Aims 1 and 3 
 
Screening 
Study Visits 1-4 
Day 1 
Day 2 
Day 3 
Day 4 
Day 5 
STUDY PROCEDURES 
 
 
 
 
 
Height 
X 
 
 
 
 
Weight 
X 
X 
X 
X 
X 


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Urine pregnancy 
 
X 
X 
X 
X 
Heart rate (electrocardiogram) 
 
X 
X 
X 
X 
Arm Blood Pressure 
X 
X 
X 
X 
X 
Core temperature 
 
X 
X 
X 
X 
Skin temperature 
 
X 
X 
X 
X 
Perceptual scales 
 
X 
X 
X 
X 
Bedside shivering scale 
 
X 
X 
X 
X 
Surface mechanomyography 
 
X 
X 
X 
X 
Resting energy expenditure 
 
X 
X 
X 
X 
International Physical Activity Questionnaire (IPAQ) 
X 
 
 
 
 
Health History Questionnaire 
X 
 
 
 
 
Health History Update Questionnaire 
 
X 
X 
X 
X 
Infrared Thermography Images 
 
X 
X 
X 
X 
PAR-Q+ 
X 
 
 
 
 
3-Day Dietary Log 
 
X 
X 
X 
X 
Body Composition 
X 
 
 
 
 
Whole Blood 
 
X 
X 
X 
X 
Fecal Samples 
 
X 
X 
X 
X 
 
 
Specific Aim 2 
 
Screening 
Study Visits 1-2 
Day 1 
Day 2 
Day 3 
STUDY PROCEDURES 
 
 
 
Height 
X 
 
 
Weight 
X 
X 
X 
Urine pregnancy 
 
X 
X 
Urine Specific Gravity 
 
X 
X 
Heart rate 
 
X 
X 
 
 
 
 
Arm Blood Pressure 
X 
X 
X 
Arterial oxygen saturation 
 
X 
X 
Partial pressure of end tidal CO2 
 
X 
X 
Ventilation 
 
X 
X 
 
 
 
 
Core temperature 
 
X 
X 
Skin temperature 
 
X 
X 
Perceptual scales 
 
X 
X 
Bedside shivering scale 
 
X 
X 
Surface mechanomyography 
 
X 
X 
International Physical Activity Questionnaire (IPAQ) 
X 
 
 
Health History Questionnaire 
X 
 
 
Health History Update Questionnaire 
 
X 
X 
Infrared Thermography Images 
 
X 
X 
PAR-Q+ 
X 
 
 
3-Day Dietary Log 
 
X 
X 
Body Composition 
X 
 
 
 
 
Specific Aim 3 
 
See table for Specific Aims 1 and 3 above. 


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9.0 
Reportable Events 
 
Safety will be constantly monitored during all data collection activities. This will occur on a 
participant-by-participant basis. If a suspected adverse event occurs in accordance with IU HRPP 
Policy on Reportable Events, the principal investigator will immediately report to the IRB and 
cooperate with the IRB in any necessary investigation. All safety data will also be reviewed in 
weekly lab meetings, particularly as it relates to changes to the risk-benefit ratio.  
 
If any of the following adverse events, which are specific to the procedures conducted in this 
study, were to occur, they will be immediately reported to the IRB using standard operating 
procedures.  
• 
Core temperature drops below 35.0°C 
• 
Infection at venous blood draw site 
• 
Loss of confidentiality 
• 
Injury requiring hospitalization and/or activation of EMS. 
• 
Death 
 
Other events deemed as events occurring outside the criteria above will be reported to the IU 
IRB reported within 5 business days using standard operating procedures. If any event occurs 
that would potentially impair the health of the participant, the experiment will be halted, and the 
IRB will be notified. 
 
Any incident, experience, or outcome that meets all the following criteria will also be reported:  
(1) unexpected (in terms of nature, severity, or frequency) given  
a. the research procedures that are described in the protocol-related documents, 
such as the IRB-approved research protocol and informed consent document; and  
b. the characteristics of the participant population being studied;  
(2) related or possibly related to participation in the research (in this guidance document, 
possibly related means there is a reasonable possibility that the incident, experience, or 
outcome may have been caused by the procedures involved in the research); and  
(3) suggests that the research places participants or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or recognized. 
 
Participants will be informed of possible risks before consenting to participate through the 
informed consent document. Details of foreseeable risks and discomforts for the procedures, and 
steps taken to lessen the probability and/or magnitude of these risks are noted below.  
 
• Acute Mirabegron ingestion: Mirabegron ingestion in the population that will be 
studied is deemed safe (3) and will be prescribed under the direction of our study 
physician, Dr. Andrew Watters. The doses that will be used in this study have mild 
cardiovascular (heart rate and blood pressure) effects that are associated with the 
dose of mirabegron (e.g., the greater the dose of mirabegron, the greater the 
cardiovascular response (2, 3). Acute oral administration of 200 mg of mirabegron has 
been shown to increase heart rate by ~8 beats per minute and systolic blood pressure 
by ~10 mmHg with no changes in diastolic blood pressure (2). These increases in 
heart rate and blood pressure are far below the heart rate and blood pressure 


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responses associated with moderate to heavy exercise intensities that are achieved in 
healthy 18-40 year old men and women.  
 
• Head Out Water Immersion: Water temperature during Specific Aim 2 will be 
perceived as being cold and rectal temperature will decrease and participants might 
shiver. We will constantly monitor rectal temperature during the study visits for 
Specific Aim 2. Any risks are reversible with removal from the cold water and warming 
with warm water perfused suits and/or mylar blankets, which are located in the 
laboratory. The protocol will be immediately terminated if core temperature drops to 
35.5°C or if the participant does not want to continue. This safety cutoff is 0.5C 
higher than the clinical definition of hypothermia and we don’t expect core 
temperature to drop to 35.5C. For instance, 60 minutes of 15C water immersion 
decreased core temperature less than 1C (8). Another study safely had participants 
sit in 10C (50F) water up to the mid-sternum for 90 minutes and core temperature 
only decreased to 36.2C (9). In a case study, 88 minutes of full body immersion in 
crushed ice lowered core temperature from 37.6C to 37.0C (10). Also, the risks 
associated with hypothermia are core body temperature-dependent and do not appear 
to be dependent on time (7).  
 
• Whole blood sample: Participants will likely experience discomfort during venous blood 
draws and in rare circumstances may feel lightheaded or faint. This risk will be 
minimized by the venipuncture always occurring in the semi-recumbent or supine 
position. There is a rare risk of infection following venous blood draws. This risk will be 
minimized by blood draws only being conducted by trained laboratory personnel. 
 
• Rectal temperature: There is an extremely low risk of rectal perforation. This risk will 
be minimized by using lubricating jelly, while rectal temperature will not be measured 
in participants with any known chronic (e.g., cancer, surgery, etc) or acute conditions 
(e.g., diarrhea, constipation, etc.) (or history of disorders) of the rectum. The 
temperature probe will be self-inserted during instrumentation processes and removed 
prior to the participant leaving the laboratory. Any risks will be reversed with removal. 
 
• Loss of confidentiality: There is a potential risk for loss of confidentiality. Every effort 
will be made to keep all subject information confidential. This risk will be alleviated by 
only using de-identified information.  
 
• Other risks: There may be other risks that we cannot predict.  
 
 
 
10.0 Data Safety Monitoring 
 
Dr. Zachary Schlader, who is independent from the study, will be responsible for data and 
safety monitoring. Dr. Schlader is an experienced environmental physiologist who specializes in 
thermoregulatory physiology. He is experienced to recognize adverse physiological responses to 
cold exposure.” Data quality, participant recruitment, accrual, retention, outcome and adverse 
event data will be monitored on a bi-weekly basis.  
 


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Data will be monitored by participant number. Thus, analysis of data will be performed without 
any identifying features to individuals. Statistical software will be used to analyze data for 
significance. Results will be reported through traditional scientific outlets such as peer-reviewed 
manuscript and presentations at national and international meetings. Communication with the 
IRB regarding safety will be reported directly by the principal investigator.  
 
11.0 Study Withdrawal/Discontinuation 
 
Participants can voluntarily withdraw from the study at any time by contacting any of the 
researchers on the study via any available means (personal contact, email, phone, etc.). If a 
researcher on the project wishes to withdraw a participant from the study, they will contact the 
participant directly (personal contact, phone, email, etc.). Possible indications for withdrawal by 
a researcher include a change in their health history that excludes them from participation and 
an inability to keep appointments or follow or understand the rules of the protocol. Data collected 
up to the time of withdraw (either voluntary or by the study team) may be used for analysis.  
 
12.0 Statistical Considerations 
 
Specific Aim 1 
Power Analysis: Using data from Loh et al. (2) we expect to observe an effect size (f) of 0.90 
for the comparison of resting energy expenditure in the placebo condition versus 200 mg of 
mirabegron. Given this effect size, 10 participants will be needed to achieve a power of 0.99 
with an alpha level set at 0.05. To account for possible attrition, we plan on recruiting up to 30 
participants. 
 
Statistical Approach: We will compare the primary (i.e., thermogenesis) and secondary (i.e., 
core temperature, skin temperature, heart rate, blood pressure, indices of shivering, thermal 
perceptions, and infrared thermography images) dependent variables using mixed effects 
ANOVA (i.e., mirabegron dose x time) followed by Tukey’s post hoc procedure if a significant 
interaction or main effect is found from the ANOVA analyses. We will also calculate the 
thermogenesis area under the curve for each dose of mirabegron. These values will be 
compared using repeated measures ANOVA followed by Tukey’s post hoc procedure if a 
significant main effect is found from the ANOVA analysis. We will also perform correlation 
analyses to determine the relation between body composition and our primary and secondary 
dependent variables. The alpha level will be set at P < 0.05 for all analyses. 
 
Specific Aim 2 
Power Analysis: Similar to Specific Aim 1 and the data from Loh et al. (2), we expect to observe 
an effect size (f) of 0.90 for the comparison of rectal temperature in the placebo condition 
versus the mirabegron condition. Given this effect size, 10 participants will be needed to 
achieve a power of 0.99 with an alpha level set at 0.05. To account for possible attrition, we 
plan on recruiting up to 30 participants. 
 
Statistical Approach: The primary (i.e., core temperature deflection point) and secondary (i.e., 
onset of shivering inflection point) dependent variables from the mirabegron and placebo trials 
will be compared using paired t-tests to determine if mirabegron improves cold-water 
immersion tolerance. Mixed effects ANOVA will be used to compare the remaining dependent 
variables between conditions (i.e., mirabegron vs. placebo) and across time. If a significant 


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interaction or main effect results from the ANOVA analyses, we will use the Holm-Sidak post 
hoc test to determine where differences exist. The alpha level will be set at P < 0.05 for all 
analyses.        
 
Specific Aim 3 
Power Analysis: This aim is being conducted as an exploratory aim in conjunction with Specific 
Aim 1. As such, to be consistent, we expect to observe an effect size (f) of 0.90 for the 
comparison of the gut microbiota in the placebo condition versus 200 mg of mirabegron. Given 
this effect size, 10 participants will be needed to achieve a power of 0.99 with an alpha level set 
at 0.05. To account for possible attrition, we plan on recruiting up to 30 participants. 
 
Statistical Approach: 
Principal Coordinate Analysis will be performed with group samples and any outliers will be 
identified. T-tests, and/or ANOVA will be performed to identify significant differences. Statistical 
analysis to detect variation between samples will be carried out using student t-test or Mann–
Whitney test depending on data distribution. Multivariate statistics will be incorporated in a 
web-based tool Metastats, which can reveal, with high confidence, discriminatory functions 
between the replicated metagenome dataset of the gut microbiota of humans. In addition, the 
ShotgunFunctionalizeR and/or Primer E packages will be used for this analysis. 
 
13.0 Statistical Data Management 
 
Primary data will be collected via a data acquisition system (PowerLab) and stored electronically 
on a password protected computer.  The storage location will be backed up manually every 
week.  Quality assurance steps will be confirmed by plotting the data prior to formal analyses. 
Outliers will be identified and checked for authenticity in the database and other original data 
documents. Extraction and cleaning of data that will be used for analysis will be carried out 
after each study visit.  
14.0 Privacy/Confidentiality Issues 
 
Informed consent will be obtained, and the testing procedures will be completed in a closed, 
private laboratory setting at the School of Public Health (SPH), with only the participant and 
investigators present.  
 
All testing will take place in a closed, private laboratory setting in the School of Public Health.  
All participants will be assigned an identification number, which will then be used when 
recording and analyzing data. A code list containing the participants names and identification 
numbers will be stored separately in a locked filing cabinet and will only be available to the 
investigators. The reports generated as a result of this investigation will not identify individual 
subjects. Data will be collected and stored on password protected computers located in the 
Human Performance Laboratories, which are always kept locked. Any paper data will be stored 
in a locked filing cabinet inside a locked office. Primary data are backed up by manual download 
to an encrypted flash drive once per week, which is then stored in a separate locked office in a 
locked filing cabinet accessible only to the investigators. 
 
While testing is in progress, a sign will be outside the laboratory door to prevent people not 
associated with the study from entering. 


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15.0 Follow-up and Record Retention 
 
We anticipate that all participants will be enrolled within 24 months. Any identifiable data (e.g., 
signed consent forms) will be maintained as required by law. De-identified data will be stored 
indefinitely. Only investigators will have full access to data. All electronic data will be kept on 
password protected computers, while paper data will remain in locked file cabinets. 
 
16.0 References 
 
1. 
Dehvari N, da Silva Junior ED, Bengtsson T, Hutchinson DS. Mirabegron: potential off 
target effects and uses beyond the bladder. Br J Pharmacol. 2018;175(21):4072-82. Epub 
2017/12/16. doi: 10.1111/bph.14121. PubMed PMID: 29243229; PMCID: PMC6177610. 
2. 
Loh RKC, Formosa MF, La Gerche A, Reutens AT, Kingwell BA, Carey AL. Acute 
metabolic and cardiovascular effects of mirabegron in healthy individuals. Diabetes Obes Metab. 
2019;21(2):276-84. Epub 2018/09/12. doi: 10.1111/dom.13516. PubMed PMID: 30203462. 
3. 
Malik M, van Gelderen EM, Lee JH, Kowalski DL, Yen M, Goldwater R, Mujais SK, 
Schaddelee MP, de Koning P, Kaibara A, Moy SS, Keirns JJ. Proarrhythmic safety of repeat 
doses of mirabegron in healthy subjects: a randomized, double-blind, placebo-, and active-
controlled thorough QT study. Clin Pharmacol Ther. 2012;92(6):696-706. Epub 2012/11/15. 
doi: 10.1038/clpt.2012.181. PubMed PMID: 23149929. 
4. 
Eltink C, Lee J, Schaddelee M, Zhang W, Kerbusch V, Meijer J, van Marle S, Grunenberg 
N, Kowalski D, Drogendijk T, Moy S, Iitsuka H, van Gelderen M, Matsushima H, Sawamoto T. 
Single dose pharmacokinetics and absolute bioavailability of mirabegron, a beta(3)-
adrenoceptor agonist for treatment of overactive bladder. Int J Clin Pharmacol Ther. 
2012;50(11):838-50. Epub 2012/09/05. doi: 10.5414/CP201782. PubMed PMID: 22943933. 
5. 
Weir JB. New methods for calculating metabolic rate with special reference to protein 
metabolism. J Physiol. 1949;109(1-2):1-9. Epub 1949/08/01. doi: 
10.1113/jphysiol.1949.sp004363. PubMed PMID: 15394301; PMCID: PMC1392602. 
6. 
Law J, Chalmers J, Morris DE, Robinson L, Budge H, Symonds ME. The use of infrared 
thermography in the measurement and characterization of brown adipose tissue activation. 
Temperature (Austin). 2018;5(2):147-61. Epub 2018/11/06. doi: 
10.1080/23328940.2017.1397085. PubMed PMID: 30393752; PMCID: PMC6209420. 
7. 
Turk EE. Hypothermia. Forensic Sci Med Pathol. 2010;6(2):106-15. Epub 2010/02/13. 
doi: 10.1007/s12024-010-9142-4. PubMed PMID: 20151230. 
8. 
Passias TC, Mekjavic IB, Eiken O. The effect of 30% nitrous oxide on thermoregulatory 
responses in humans during hypothermia. Anesthesiology. 1992;76(4):550-9. Epub 1992/04/01. 
doi: 10.1097/00000542-199204000-00011. PubMed PMID: 1550280. 
9. 
Rostomily KA, Jones DM, Pautz CM, Ito DW, Buono MJ. Haemoconcentration, not 
decreased blood temperature, increases blood viscosity during cold water immersion. Diving 
Hyperb Med. 2020;50(1):24-7. Epub 2020/03/19. doi: 10.28920/dhm50.1.24-27. PubMed PMID: 
32187614; PMCID: PMC7276274. 
10. 
Bongers C, Eijsvogels TMH, Thijssen DHJ, Hopman MTE. Thermoregulatory, metabolic, 
and cardiovascular responses during 88 min of full-body ice immersion - A case study. Physiol 
Rep. 2019;7(24):e14304. Epub 2019/12/29. doi: 10.14814/phy2.14304. PubMed PMID: 
31883220; PMCID: PMC6934874. 
 
17.0 Appendix 


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17.1 Payment schedule 
Specific Aim 1: Participants will be compensated in the form of a gift card for 
$495 for completing the informed consent/screening visit and the four study 
visits. Compensation will occur on or after the final visit to the laboratory. If 
participants do not complete the study, they will be compensated for their time 
spent in the laboratory at a rate of $15/hour paid by gift card. 
 
Specific Aim 2: Participants will be compensated in the form of a gift card for 
$165 for completing the informed consent/screening visit and both study visits. 
Compensation will occur on or after the final visit to the laboratory. If 
participants do not complete the study, they will be compensated for their time 
spent in the laboratory at a rate of $15/hour paid by gift card. 
 
Specific Aim 3: Participants will be compensated in the form of a gift card for 
$100 for completing the informed consent/screening visit and brining their 
samples to the laboratory. Compensation will occur on or after the final visit to 
the laboratory. If participants do not complete the study, they will be 
compensated at a rate of $25/sample paid by gift card. 
 
 
17.2 Attachments  
The following Questionnaires have been included as attachments:  
o International Physical Activity Questionnaire (IPAQ) 
o Physical Activity Readiness Questionnaire Plus (PAR-Q+) 
o Healthy History and Demographics Questionnaire 
o Health History Update Questionnaire  
 
The following Recruitment tools have also been included as attachments:  
o Recruitment flyer  
o Email script  
o Verbal script  
o Phone script 
 

ICF_001.pdf: 
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Mirabegron and Physiological Function in Cold Environments 
 
NCT04766021 
 
January 29, 2024 
 


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        Page 2 of 11 
INDIANA UNIVERSITY INFORMED CONSENT STATEMENT FOR RESEARCH 
Mirabegron and Physiological Function in Cold Environments 
Mirabegron and Thermogenesis in Cold Air - Funded by the Office of Naval Research 
 
ABOUT THIS RESEARCH  
You are being asked to participate in a research study.  Scientists do research to answer important 
questions which might help change or improve the way we do things in the future.  
 
This consent form will give you information about the study to help you decide whether you want to 
participate.  Please read this form, and ask any questions you have, before agreeing to be in the study. 
 
TAKING PART IN THIS STUDY IS VOLUNTARY 
You may choose not to take part in the study or may choose to leave the study at any time.  Deciding not to 
participate, or deciding to leave the study later, will not affect your grades or result in any penalty or loss of 
benefits to which you are entitled, and will not affect your relationship with the Indiana University – 
Bloomington School of Public Health, or the Human Performance Laboratories. 
 
Study Summary 
Navy Divers often perform work in cold water. However, their protective equipment is cumbersome and 
does not completely prevent decreases in body temperature during cold water dives. Thus, alternative 
solutions that keep divers warm during cold water exposure are needed to improve diver safety.  
 
Mirabegron is an FDA approved drug for the treatment of overactive bladder. Mirabegron also increases 
body temperature and therefore might be an alternative strategy that can improve cold water tolerance in 
divers. However, before we examine the effects of mirabegron during cold water immersion we need to 
know which dose of mirabegron increases body temperature the most. Therefore, the purpose of this study 
is to determine which dose of mirabegron can elevate body temperature the most during mild cold air 
exposure.  
 
You will be asked to come to the laboratory for five visits (~33 total hours); one screening visit (~1 hour) 
and 4 study visits (~8 hours each). During the screening visit we will determine your eligibility, resting blood 
pressure and heart rate, and determine your body composition. During the 4 study visits, you will ingest a 
single dose of mirabegron or a placebo (a sugar pill) and spend 6 hours in a room that is mildly cold.  We 
will measure your body temperature, heart rate, blood pressure, and shivering during the study. We don’t 
expect you to receive any benefit from taking part in this study, but we hope to learn things which will help 
scientists in the future. Taking part in this research may expose you to risks. These risks include tearing of 
the rectum, infection, and feeling cold. We will attempt to minimize these risks by having all procedures 
performed by trained individuals and through monitoring of your condition during the study. Payment for 
your time is available if you decide to take part in this study. There is no cost to you for taking part in this 
study.  
 


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Please review the rest of this document for more details about this study and the things you should know 
before making a decision about whether to participate in this study. 
 
WHY IS THIS STUDY BEING DONE? 
Navy and Special Warfare Divers are often exposed to cold water during their missions. Although these 
divers wear thermal protection, it is often cumbersome and does not completely protect the diver from the 
cold. This can affect the diver’s performance and safety while trying to carry out their tasks. Therefore, new 
strategies are needed to improve tolerance to cold water. Mirabegron is an FDA-approved drug for the 
treatment of overactive bladder. Mirabegron also raises body temperature. Therefore, divers could ingest 
mirabegron prior to a cold water dive in order to increase their body temperature and improve cold water 
tolerance. However, we don’t know what dose of mirabegron can increase body temperature for extended 
periods of time. Therefore, the purpose of this study is to determine which dose of mirabegron can elevate 
body temperature the most during mild cold air exposure. 
 
You were selected as a possible participant because you are 18-40 years of age and generally healthy.    
 
The study is being conducted by Dr. Blair Johnson and the Indiana University – Bloomington, Department of 
Kinesiology within the School of Public Health. It is funded by a grant from the Office of Naval Research.   
 
HOW MANY PEOPLE WILL TAKE PART? 
If you agree to participate, you will be one of 30 participants taking part in this research.  
 
WHAT WILL HAPPEN DURING THE STUDY? 
If you agree to be in the study, you will do the following things: 
• You will be asked to visit the Human Performance Laboratories at the School of Public Health 
Building at 1025 E. 7th Street, Room 075, Bloomington, IN 47405. Parking is free in designated 
parking spots behind the building or in the parking lot out front. 
• For these visits you will be asked to wear comfortable clothing during the visit at the laboratory 
(men: no shirt and shorts; women: sports bra and shorts) (visits 2-5). 
• We expect that you will be in this research study for about 33 hours over a total of five visits. The 
first visit will be used for informed consent (about 1 hour) and the remaining four visits will be used 
for the study visits (about 8 hours each).  Each study visit will be separated by at least 10 days. If you 
are determined not to be eligible based on screening, you will be withdrawn after the first visit. 
Please consider your schedule and any classes or meetings that may be affected by the strict time 
requirements for this study. 
 
This is a brief description of what will happen on each study visit.  There are further details below this 
section explaining each study procedure as well as the risks associated with that procedure. 
 
• Screening / Visit 1:  Once you arrive at the laboratory, an investigator will get written informed 
consent after all procedures and study risks are fully explained and all questions answered. After 


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obtaining written informed consent, you will complete a Health History and Demographics 
Questionnaire, an International Physical Activity Questionnaire (IPAQ), and a Physical Activity 
Readiness Questionnaire (PAR-Q+). An experienced investigator will administer these 
questionnaires and they can fully explain any technical terms and answer any inquiries related to 
the questionnaires. Your blood pressure and heart rate will be measured. You will meet with our 
study physician to determine if you should not take mirabegron or participate in the study. You will 
be taught how to use the myfitnesspal dietary log (myfitnesspal.com or mobile app). Prior to each 
remaining study visit, you will show the study staff your food and fluid intake for the 3 days before 
your study visit. You will also have your height and weight measured and your body composition will 
be assessed (dual-energy X-ray absorptiometry (DEXA)). During the DEXA scan, you will lay on your 
back for approximately 10 minutes while the scanner images your body to determine your body 
composition. If you cannot see our physician or perform the body composition assessment during 
this visit, additional visits can be scheduled. You can also visit with the study physician over a Zoom 
call to review your medical history. The total time for this screening visit will take approximately 1 
hour. 
 
• Visits 2-5:  You will be asked to complete 4 visits that will take approximately 8 hours each to 
complete. For 3 days prior to each study visit, you will be asked to record the amount and type of 
food and fluid that you consume using myfitnesspal dietary log. This free program is available at the 
website (myfitnesspal.com) or as an app for your mobile device. We ask that you consume the same 
amount and type of food and fluid for 3 days prior to each visit because your diet might influence 
the results of your study. We will view your dietary log to make sure that you consume the same 
type and amount of food and fluid prior to each visit. You will be asked to report to the laboratory in 
the morning after no exercise, caffeine, and alcohol for 24 hours, and no food for 12 hours. You will 
also be asked to not take any medication the day of these visits (except birth control). Men will be 
asked to be shirtless and wear shorts and women will be asked to wear a sports bra and shorts 
during these visits.  
 
• These 4 visits will only differ by the amount of mirabegron or placebo that you will ingest. The order 
of these visits will be randomly determined (by chance, like rolling a die).  
▪ Placebo 
▪ 100 mg mirabegron 
▪ 150 mg mirabegron 
▪ 200 mg mirabegron 
 
• Upon arrival at the laboratory, you will complete a Health History Update Questionnaire to ensure 
no relevant changes in your health history have occurred. Women of child-bearing potential will 
give a urine sample that will be used for a urine pregnancy test. The remaining urine may be stored 
for later analyses. You will consume a light breakfast (237 ml (8 fluid oz) of Ensure Original; 220 
kcal). You will then self-insert a rectal probe that will be used to measure your internal body 
temperature.  


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• We will attach equipment to monitor you over the course of the study. There will be various tests 
that are described in detail below under “Experimental Procedures”.  
 
• After 20 minutes of quiet resting, a baseline set of recordings will be started and a small amount of 
blood (approximately 3 teaspoons or 15 mL) will be taken from a vein in your arm before you ingest 
the mirabegron or placebo  
 
• We will then have you ingest the mirabegron or placebo and 30 minutes following ingestion, you 
will enter the whole-body indirect calorimeter room. This room is designed to comfortably measure 
your energy expenditure by collecting the air you breathe out over a long period of time.  The 
temperature of the room will be set to 68F (20C). 
 
• You will spend 6 hours in the whole-body indirect calorimeter room. During the 6 hours, you will be 
free to watch television or read. We will ask that you perform the same amount of television 
watching and reading between study visits. For instance, if you watch television for 3 hours during 
one visit, you will be asked to watch television for 3 hours during subsequent visits. 
 
• Every 30 minutes we will record your body temperature, heart rate, and blood pressure. 
 
• Every 60 minutes you will be asked to lay down and rest for 30 minutes as we record your energy 
expenditure. 
 
• You will be given the opportunity to use the restroom after approximately 3 hours in the chamber 
and urine will be collected at this time. 
 
• At the end of the 6 hour protocol, you will exit the whole-body indirect calorimeter and we will 
record your body temperature, heart rate, and blood pressure and a small amount of blood 
(approximately 3 teaspoons or 15 mL) from a vein in your arm. After this we will give you a blanket 
to warm yourself if you choose.  
 
• Prior to leaving the laboratory, we will provide you with a fecal (stool) collection kit. We will give 
you verbal and written instructions on how to obtain a small fecal sample (less than 1 teaspoon or 
0.5 mL) from your first bowel movement following the study visit. We will provide you with shipping 
instructions or you can bring the sample back to the laboratory and a staff member will ship the 
sample for analysis. 
 
Experimental Procedures: The procedures listed below are designed for research and are not for medical 
purposes. 
 
 
 


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Mirabegron: 
Description: You will ingest mirabegron tablets with a small glass of water. The dose of mirabegron, 100 
mg, 150 mg, and 200 mg will be randomly selected for each study visit. 
Potential Risks: There are risks associated with ingesting mirabegron such as, increased blood pressure and 
heart rate, nasopharyngitis (common cold), urinary tract infection, urinary retention, angioedema (swelling 
in the skin or lips), headache, constipation, upper respiratory tract infection, arthralgia (joint pain), 
diarrhea, abdominal pain, and fatigue.  
Duration: Once for each study visit. 
 
DEXA Scan: 
Description: You will lay on your back while the scanner images your body to determine your body 
composition. 
Potential Risks: If you have had radiation (like x-rays, CT or radiation therapy) before or you participated in 
a different study where you were exposed to radiation, please tell us now. We want to make certain that 
the probability of harm from the amount of radiation you will be exposed to in this study continues to be 
low when combined with the radiation you have received within the past year. If you are pregnant, you 
cannot take part in this research study. If you are able to have a baby, are not pregnant and wish to take 
part in this study, you will need to take a pregnancy test prior to enrollment. If you get pregnant while 
taking part in this study, or think you are pregnant, please tell the research team right away. You will not be 
able to continue this study if you become pregnant. 
 
The DEXA scan that you get in this study will expose you to low amounts of radiation.  Every day, people are 
exposed to low levels of radiation that come from the natural environment and man-made radiation 
sources around them.  This type of radiation is called “background radiation.” The amount of radiation you 
will get from the scans in this study is approximately, or less than, 1 years’ worth of background radiation.  
The probability of harm from participating in this study is low compared to other everyday risks. Certain 
diseases or conditions may affect your sensitivity to radiation. For more detailed information on the risks of 
radiation or if you wish to have a more detailed dose estimate, please ask the research team involved with 
this study. 
 
For women of child-bearing potential: This study may be hazardous to an unborn child. The amount of 
radiation exposure necessary to present a significant risk to an unborn child is not well established; 
however, recommendations exist which limit the exposure to the unborn child of individuals who are 
occupationally exposed to radiation (e.g. x-ray technologists, radiologists, etc.) to no more than 500 
millirem (mrem). Should you be pregnant and participate in this study, the radiation dose to the unborn 
child would be less than 500 mrem; however, pregnant individuals should not participate in this study. To 
assure that you are not pregnant, a pregnancy test will be performed at no cost to you, prior to initiation of 
the study. The results of the pregnancy test will be made available to you before the study is initiated. 
Furthermore, adequate birth control measures should be taken during your participation in this study. 
Duration: Once, for approximately 5-10 minutes during Visit 1. 
 
 


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Electrocardiogram (measure of the electrical output of your heart): 
Description: Sticky patches will be applied to your skin to measure heart rate from your heart’s electrical 
signals. 
Potential Risks: There may be some discomfort from removing the tape. 
Duration: During all visits except Visit 1, continually measured.  
 
Skin Temperature: 
Description: Sticky patches will be applied to your skin to measure your skin temperature. 
Potential Risks: There may be some discomfort and/or redness from removing the tape. 
Duration: During all visits except Visit 1, continually measured.  
 
Arm Blood Pressure: 
Description: Your blood pressure and heart rate will be monitored using a cuff placed on your upper arm 
that is inflated and deflated periodically. 
Potential Risks: You may feel slightly uncomfortable due to inflation of the cuff around your arm, but there 
are no risks. 
Duration: The cuff will be on your upper arm during all visits.  Blood pressure measurements will be taken 
at several different time points. Each measurement will last approximately 30 seconds. 
 
Infrared Thermography Images: 
Description: We will take pictures of your upper body to measure skin temperature near your collar bone. 
Potential Risks: None. 
Duration: During all visits except Visit 1, every 30 minutes. 
 
Surface Mechanomyography (shivering): 
Description: We will adhere triaxial accelerometers, similar to a watch, to your chest, upper back, and thigh 
using tape. 
Potential Risks: There may be some discomfort from removing the tape. 
Duration: During all visits except Visit 1, continually measured. 
 
Bedside Shivering Scale: 
Description: Two investigators will rate how much you are shivering. 
Potential Risks: None. 
Duration: During all visits except Visit 1, every 30 minutes. 
 
Perceptual Scales (questionnaires):  
Description: You will be shown standardized scales to rate your whole-body thermal sensation 1 (“cold”) to 
7 (“hot”) and thermal comfort 1 (“comfortable”) to 4 (“very uncomfortable”)  
Potential Risks: None.  
Duration: During all visits except Visit 1, every 30 minutes. 
 


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Energy Expenditure (thermogenesis):   
Description: The air you breathe out will be used to calculate energy expenditure.   
Potential Risks: None. 
Duration: During all visits except Visit 1, every 60 minutes. 
 
Urine Sample: 
Description: You will provide urine samples in a private bathroom that will be tested for pregnancy status 
(women of childbearing potential only) and urine specific gravity for both men and women.  
Potential Risks: None. 
Duration: During all visits except Visit 1, before we attach you to our monitoring devices, after ~3 hours in 
the chamber, and at the end of each study visit.  
 
Body Height and Weight: 
Description: You will be asked to measure your height and weight without clothes on using a stadiometer 
(i.e., a scale that is used to measured height and weight) situated in a private room.  
Potential Risks: None. 
Duration: During all visits, before we attach you to our monitoring devices.   
 
Rectal Temperature 
Description: In order to estimate internal body temperature, you will self-insert a small (~3mm/0.118 
inches in diameter), lubricated, single-use rectal temperature probe about 4 inches. You will insert and 
remove the probe in a private room.  
Potential Risks: Insertion of the temperature probe should not be uncomfortable or painful. There is a very 
slight risk that you will perforate your rectum. To minimize this risk the temperature probe will be 
lubricated. Furthermore, rectal temperature will not be measured if you have (or have had) any chronic 
disorders of the rectum (e.g., cancer, surgery etc.) or if you currently have any related acute conditions 
(e.g., diarrhea, constipation, etc.).  
Duration: During all visits except Visit 1, continually measured.  
 
Blood Sample 
Description: A small amount of blood (~3 teaspoons or 15 mL) will be taken from an arm in your vein using 
aseptic techniques that are guided by the World Health Organization.  A sample of your blood will be 
shipped to our collaborator at Rutgers University for analysis. 
Potential Risks: There is a small risk of infection at the site. This will be minimized by using trained 
personnel who will use aseptic techniques and gloves to properly clean and cover the blood draw site with 
a bandage.  
Duration: Two samples during each visit except Visit 1. A total of ~24 teaspoons (or 120 mL) will taken over 
the entire study (~6 teaspoons or 30 mL per study visit). 
 
 
 


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Fecal Sample 
Description: You will be given a fecal sample collection kit (DNA Genotek) to take home with you. You will 
be asked to take a small sample (less than 1 teaspoon or 0.5 mL) of your feces (stool) from your first bowel 
movement after each study visit following the provided directions. A sample of your feces will be shipped 
(either by you or a lab member) to our collaborator at Rutgers University for analysis. 
Potential Risks: None. 
Duration: One sample after study visits 2-5 (4 total samples, less than 4 teaspoons total). 
 
WHAT ARE THE RISKS OF TAKING PART IN THE STUDY? 
While participating in the study, the risks, side effects, and/or discomforts include: 
Procedures:  
Because of your participation in this study, you are at risk for the aforementioned side effects (see Potential 
Risks above).  
 
Loss of Confidentiality 
Any time information is collected there is a potential risk for loss of confidentiality. Every effort will be 
made to keep your information confidential. However, this cannot be guaranteed. 
  
Other Risks 
There also may be other side effects that we cannot predict.  
 
 
WHAT ARE THE POTENTIAL BENEFITS OF TAKING PART IN THE STUDY? 
There will be no direct benefit to you from taking part in this research study. However, we hope that the 
information learned from this study will provide new information regarding the control of hemodynamics 
and ventilation during water immersion.  
 
WILL I RECEIVE MY RESULTS? 
We may learn things about you from the study activities, but these results are considered research and will 
not be returned to you. 
 
HOW WILL MY INFORMATION BE PROTECTED? 
Efforts will be made to keep your personal information confidential.  We cannot guarantee absolute 
confidentiality.  Your personal information may be disclosed if required by law.  No information which could 
identify you will be shared in publications about this study. 
 
Organizations that may inspect and/or copy your research records for quality assurance and data analysis 
include groups such as the study investigator and his/her research associates, the Indiana University 
Institutional Review Board or its designees, and any state or federal agencies who may need to access your 
medical and/or research records (as allowed by law).  State and federal agencies may include the Office for 
Human Research Protections (OHRP), the US Office of Naval Research Human Research Protection Office, 
and the Food and Drug Administration (FDA). 


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A description of this clinical trial will be available on ClinicalTrials.gov as required by U.S. Law. This Web site 
will not include information that can identify you. At most, the Web site will include a summary of the 
results. You can search this Web site at any time. 
 
WILL MY INFORMATION BE USED FOR RESEARCH IN THE FUTURE? 
Information or specimens collected from you for this study may be used for future research studies or 
shared with other researchers for future research.  If this happens, information which could identify you 
will be removed before any information or specimens are shared.  Since identifying information will be 
removed, we will not ask for your additional consent.  Your samples will not be used for genetic testing. We 
will not use the specimens collected as a part of this study for whole genome sequencing, which involves 
mapping all of your DNA. 
 
WILL I BE PAID FOR PARTICIPATION?  
If you agree to take part in this research study, we will compensate you $495 for completing visits 1-5 and 
$100 for submitting all 4 fecal samples. Money will be paid when you complete or choose to quit the study 
and will be paid by gift card. If you do not complete the study, you will be compensated only for your time 
in the study ($15/hour) and $25 for each fecal sample. 
 
If you receive $600 or more in one calendar year from Indiana University, you will be required to provide 
your Social Security number or tax identification number to Indiana University.  You will receive a 1099 tax 
form the following January and will need to report this payment as income on your federal and state tax 
returns.  You are responsible for paying any state, federal, or Social Security taxes.  If you have questions 
regarding how this impacts your tax return, please contact a tax professional to assist you.  If you do not 
have a social security number or tax identification number, the Internal Revenue Service (IRS) requires 
Indiana University to deduct 30% from your compensation to pay required taxes on your behalf. 
 
WILL IT COST ME ANYTHING TO PARTICIPATE?  
There is no cost to you for taking part in this study.  
 
WHO WILL PAY FOR MY TREATMENT IF I AM INJURED?  
In the event of physical injury resulting from your participation in this study, necessary medical treatment 
will be provided to you and billed as part of your medical expenses.  Costs not covered by your health care 
insurer will be your responsibility.  Also, it is your responsibility to determine the extent of your health care 
coverage.  There is no program in place for other monetary compensation for such injuries.  However, you 
are not giving up any legal rights or benefits to which you are otherwise entitled.   
 
If you are participating in research that is not conducted at a medical facility, you will be responsible for 
seeking medical care and for the expenses associated with any care received.   
 
 
 


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WHO SHOULD I CALL WITH QUESTIONS OR PROBLEMS? 
For questions about the study or a research-related injury, contact the researcher, Dr. Blair Johnson at 812-
855-8699.  After business hours or in the event of an emergency, please call Dr. Blair Johnson at the same 
phone number.   
 
For questions about your rights as a research participant, to discuss problems, complaints, or concerns 
about a research study, or to obtain information or to offer input, please contact the IU Human Subjects 
Office at 800-696-2949 or at irb@iu.edu. 
 
CAN I WITHDRAW FROM THE STUDY? 
If you decide to participate in this study, you can change your mind and decide to leave the study at any time in the 
future.  The study team will help you withdraw from the study safely.  If you decide to withdraw, please notify Dr. 
Johnson of your decision. Withdrawing will not result in any penalty or loss of benefits to which you are entitled 
and will not affect your relationship with the Indiana University – Bloomington School of Public Health, or 
the Human Performance Laboratories. 
 
Your participation may be terminated by the investigator without regard to your consent in the following 
circumstances: your inability to follow direction, if you cannot attend your scheduled appointments, or if a 
change in your health history between any of the visits to the laboratory meet any of the exclusion criteria.  
 
 
PARTICIPANT’S CONSENT 
In consideration of all of the above, I give my consent to participate in this research study.  I will be given a 
copy of this informed consent document to keep for my records.  I agree to take part in this study. 
 
Participant’s Printed Name: 
 
 
Participant’s Signature: 
Date: 
 
 
Printed Name of Person Obtaining Consent:  
 
 
 
Signature of Person Obtaining Consent: 
Date: