Exercise Training as an Intervention to Improve Muscle Function and Recovery Following Bed Rest in Older Adults With Type 2 Diabetes
Phase: NA
Conditions: Type 2 Diabetes, Muscular Atrophy, Sedentary Behavior, Exercise, Pre-diabetes
Sponsor: AdventHealth Translational Research Institute

 
 
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Study Protocol Title: 
Exercise training as an intervention to improve muscle function and recovery following bed rest 
in older adults with type 2 diabetes (RECOVER) 
 
Study Sponsor:   
AdventHealth Orlando 
 
Principal Investigator: 
Paul M. Coen, Ph.D. 
 
Medical Investigator: 
Richard Pratley, M.D. 
 

 
 
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Table of Contents: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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List of Abbreviations: 
 
AE - Adverse Event 
AF - Activity Factor 
AH – AdventHealth 
AH TRI – AdventHealth Translational Research Institute 
AMP - Adenosine Monophosphate-Activated Protein  
ATP - Adenosine Triphosphate 
BMI - Body Mass Index 
BP - Blood Pressure 
BR - Bed Rest 
BUN - Blood Urea Nitrogen 
CaHMB - Calcium-Beta-Hydroxy-Betamethylbutyrate 
CON – Ambulatory (Non-Exercised) Control Group 
CRF - Case Report Form 
CRU - Clinical Research Unit 
DBP - Diastolic Blood Pressure 
DEXA - Dual-Energy X-ray Absorptiometry 
DNA - Deoxyribonucleic Acid 
DO - Doctor of Osteopathic Medicine 
DPP - Dipeptidyl Peptidase 
DSMB - Data Safety Monitoring Board 
DVT - Deep Vein Thrombosis  
ECG - Electrocardiogram 
EGP - Endogenous Glucose Production 
EX – Exercise Intervention Group 
FDA - Food and Drug Administration 
FFA - Free Fatty Acid 
eGFR - Estimated Glomerular Filtration Rate 
GDR - Glucose Disposal Rate  
GLP - Glucose-Like Peptide 
HbA1c - Hemoglobin A1c 
HDL - High-Density Lipoprotein 
HFD - High Fat Diet 
HIPAA - Health Insurance Portability and Accountability Act authorization 
HIT - Heparin Induced Thrombocytopenia  
HIV - Human Immunodeficiency Virus 
HR - Heart Rate 
HRT - Hormone Replacement Therapy 
HsCRP – high sensitivity C-Reactive Protein 
ICF - Informed Consent Form 
IMCL - Intramyocellular Lipid 

 
 
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IR - Insulin Resistance 
IV - Intravenous  
LDL - Low-Density Lipoprotein 
LMWH - Low Molecular Weight Heparin 
MD - Medical Doctor 
MRI - Magnetic Resonance Imaging 
MRS - Magnetic Resonance Spectroscopy 
NP - Nurse Practitioner  
PA - Physician Assistant 
PBR - Pre-Bed Rest 
PCP - Primary Care Physician 
PE - Pulmonary Embolism 
PI - Principal Investigator 
PID - Participant Identification Number 
QD - Per Day 
REE - Resting Energy Expenditure 
RER - Respiratory Exchange Ratio 
RM - Repetition Maximum 
RMR - Resting Metabolic Rate 
ROM - Range of Motion 
ROS - Reactive Oxygen Species 
RQ - Respiratory Quotient 
SAE - Serious Adverse Event 
SAS - Statistical Analysis System 
SBP - Systolic Blood Pressure 
SDH - Sorbitol Dehydrogenase 
SI - Insulin Sensitivity 
SOP - Standard Operating Procedures 
SPPB - Short Performance Physical Battery 
T2D - Type 2 Diabetic 
TRI - Translational Research Institute  
TSH - Thyroid Stimulating Hormone 
VO2max - Maximal Oxygen Uptake 
VTE - Venous Thromboembolism 
400MWT - 400-Meter Walk Test 
 
 

 
 
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Introduction 
 
This document is a protocol for a human research study. This study is to be conducted in 
accordance with applicable Federal regulations and institutional research policies and procedures. 
 
Older adults with type 2 diabetes (T2D) experience an accelerated rate of sarcopenia, which is the 
deterioration in muscle mass, strength, and physical performance. Periods of disuse caused by 
illness or hospitalization causes rapid loss of muscle mass and strength, which negatively impact 
physical function upon re-ambulation. In fact, without adequate rehabilitation, physical function 
does not fully recover in older adults following disuse, reducing the quality of life and loss of 
independence 
 
There is a lack of effective therapeutic options to aid muscle recovery following disuse, in part, 
because the myocellular mechanisms underlying the atrophy and recovery programs in human 
muscle are not well characterized. Our long-term goal is to identify specific molecular targets for 
the development of evidence-based clinical interventions to counteract the negative health 
outcomes associated with muscle atrophy induced by illness, injury, or hospitalization. Our overall 
objectives are to employ highly innovative methods in muscle biopsy specimens in order to 
decipher the temporal sequence by which mitochondrial dysfunction and lipotoxicity in older 
adults with T2D impact atrophy and recovery of muscle mass, strength and physical function 
following bed rest. Studies in the clinically relevant context of aging, T2D and exercise will 
provide a physiological framework in which the temporal and mechanistic relationships between 
mitochondria, intramyocellular lipid and protein synthesis/breakdown can be interrogated during 
atrophy and recovery. 
 
Our primary approach is to conduct a prospective, parallel two arm, randomized controlled study 
consisting of five discrete but connected phases: 
 
I) Screening, II) Pre-Bed Rest, III) Bed Rest, IV) Ambulatory/Exercise Recovery and V) 
Resistance Exercise (OPTIONAL) 
 
Eligible participants will be Older Adults with T2D and will be randomly assigned to one of two 
groups:  Exercise recovery (EX) or Ambulatory recovery (CON).  
 
The expected duration for a subject in this study could be up to 16 weeks. 
 
A series of physiologic and clinical/health outcomes will be assessed before, during and after the 
bed rest intervention period and recovery period: 
1) Morning resting fasted variables: blood pressure; blood glucose and lipids. 2) Physical 
Examination, Resting EKG, Urine collection (for urinalysis, BUN, creatinine). 3) 
Cardiorespiratory fitness (VO2peak) determined via a graded cycle ergometry test. 4) Body 
composition by; DXA scan - bone mineral density, total and partitioned lean mass, estimated 
visceral fat; and MRI – left and right leg mid-thigh muscle, bone and fat volume and area. 5) 
Muscle strength and power: Quadriceps muscle performance testing (maximal strength and power) 

 
 
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will be performed using a Biodex dynamometer. 6)Physical function: The Short physical 
performance battery (SPPB) will be used to measure lower extremity performance as a secondary 
outcome. 7) MRS: Mitochondrial energetic capacity (ATPmax) will be determined by 31P MRS with 
a brief (24 sec) bout of isometric exercise protocol. 8) Hyperinsulinemic euglycemic clamp to 
determine insulin sensitivity. 
Participants will enter a 10-day strictly controlled bed rest intervention at the Translational 
Research Institute (TRI) clinical research unit and a progressive 4-week aerobic and resistance 
training recovery program at the exercise training facility at the TRI, if not randomized to the 
ambulatory recovery group. Both groups can also receive additional optional resistance exercise 
training following 4 weeks of the ambulatory or exercise recovery phase. Blood and skeletal 
muscle biopsies will be collected immediately followings days 10 of the bed rest period and after 
1, 2, 3 and 4 weeks of the recovery period. Adipose tissue biopsies will also be collected on bed 
rest day one, immediately following 10 days of bed rest, and after 4 weeks of recovery if the 
participant elects to do this. The adipose biopsy is available to be chosen by the participant but not 
required. 
 
Background Information and Scientific Rationale 
 
Premise: Skeletal muscle atrophy is a clinically significant problem that occurs during disuse 
or immobilization due to hospitalization, illness and injury, and leads to a loss of muscle strength 
and physical function (28, 42, 66). This is a particular public health problem for older adults who 
comprise the majority of hospital patients in the US (26, 38) and who may lose more muscle mass 
during bed rest (64, 88). 
Older adults do not adequately recover following bed rest without adequate rehabilitation (57, 
112, 113), which likely contributes to their reduced functional status and ambulation upon 
discharge (20, 22), a loss of independence, nursing home placement (39) and an increased risk for 
falls and fractures (80). Older adults are also more likely to develop type 2 diabetes (T2D). Indeed, 
~33% of all hospital patients in the US have T2D. Evidence from the longitudinal Health ABC 
study showed that T2D is associated with more rapid loss of muscle mass and strength in older 
adults (89). Furthermore, older adults with T2D are at increased risk of injurious falls (78) and 
physical disability (25). Despite the potentially serious clinical implications however, T2D has not 
been investigated as an important factor in muscle atrophy and recovery response with 
immobilization.  
Mechanisms underlying disuse atrophy and poor muscle recovery in humans are not well 
characterized. Most immobilization studies in humans have been limited to measuring changes 
in muscle only before and after disuse atrophy and recovery (20, 21, 64, 65, 113, 114). This has 
contributed to discordant views in the field regarding the causal roles that deranged protein 
synthesis or degradation play in atrophy responses (43, 94, 96). Supported by evidence from 
preclinical rodent models (8), clinical investigations of early time course changes during atrophy 
and recovery responses could reveal temporal mechanistic relationships, leading to identification 
of muscle targets in humans. For example, only 24-48 hrs. of inactivity can reduce muscle protein 
synthesis (115), and reduce phosphorylation of AKT, a key upstream regulator of mTOR/p70S6K 
(111, 118). 

 
 
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The role of mitochondrial energetics in disuse atrophy in humans is unknown. The metabolic 
correlates to muscle atrophy include skeletal muscle insulin resistance (21, 23, 75), decreased fatty 
acid oxidation and a shift to glucose oxidation (9), intramyocellular lipid accumulation (23, 110) 
and impaired protein synthesis (36, 42, 87). While mitochondria have been implicated in the 
etiology of these metabolic dysfunctions in the context of aging and diabetes (44, 92), the role of 
mitochondria in human muscle disuse atrophy and loss of function is poorly understood.   
Mitochondrial reactive oxygen species as an upstream mediator of impaired protein 
synthesis. Release of pro-apoptotic factors (1, 73) morphological alterations (fission, swelling), 
energy stress via reduced ATP (100), and increased mitochondrial reactive oxygen species (ROS) 
emission (1, 60, 79, 82) have all been reported during muscle atrophy in pre-clinical studies. It is 
not clear whether these alterations in muscle mitochondria are simply a consequence of myofiber 
atrophy or actively contribute to muscle atrophy. Pre-clinical evidence indicates that mitochondrial 
ROS can depress protein synthesis by decreasing phosphorylation of 4E-BP1 and impairing mTOR 
assembly (93, 104, 120) and also induce muscle insulin resistance in part by impairing postprandial 
AKT phosphorylation, a major driver of the mTOR pathway and protein synthesis (5).  
Mitochondria activate protein degradation systems. Mitochondria-targeted antioxidant 
treatment in rodents support a crucial role for mitochondrial ROS in mediating muscle atrophy 
(79, 95). Increased ROS production can also exacerbate muscle atrophy (59). Mitochondrial ROS 
stimulate proteolytic degradation pathways (autophagy & proteasome system) (7, 24, 49, 55, 70, 
74, 106) and energetic stress (reduced ATP production), which can activate the AMP kinase 
(AMPK)-FoxO3 pathways leading to increased expression of ubiquitin-proteasome system and 
lysosome-autophagy system (48, 100). Taken together, multiple lines of compelling pre-clinical 
evidence, including data from our group presented later, implicate a central role for mitochondrial 
energetics in muscle atrophy (Fig. 1). A handful of small, underpowered human studies provide 
circumstantial evidence that mitochondrial dysfunction occurs during limb immobilization (11, 
46, 47, 111) but none have adequately interrogated a potential role for muscle mitochondria 
during disuse atrophy and recovery. 
The mechanisms by which intramyocellular lipids contribute to human muscle atrophy is 
unknown. Evidence from both pre-clinical models and humans suggest that impaired 
mitochondrial fatty acid oxidation (41) and intramyocellular lipid (IMCL) may play a role in 
muscle atrophy (23). Specific lipotoxic species including ceramide are associated with poor 
anabolic response to exercise in older adults (99). High fat diet (HFD) fed rodents and muscle cell 
culture models consistently link ceramide with exacerbated muscle atrophy or impaired anabolic 
signaling (58, 72, 98, 101, 108). Excess intramyocellular lipids are also linked with poor muscle 
quality, and with slower myofiber contraction, force and power development in obese older adults 
(13, 45). The role of IMCL in general and ceramides specifically in human muscle atrophy has not 
been examined. 
T2D may be a critical factor linking mitochondrial energetics, imcl and disuse atrophy. Older 
adults are becoming more obese along with having an increased incidence of T2D. Findings from 
our group and others indicate that skeletal muscle insulin resistance in T2D is mediated by lipid 
induced mitochondrial stress (83). T2D, Obesity and IR have been shown to contribute to blunted 
control of sub-fraction specific-protein synthesis by insulin, amino acids and other anabolic stimuli 
(12, 50-53, 84, 86). Although mechanistic studies are limited, studies reporting reduced muscle 

 
 
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protein synthesis by experimental elevation of circulating fatty acids (85, 108) support a putative 
connection between IR and an exacerbated atrophy response. These impairments could also impact 
functional recovery following periods of inactivity. Moreover, we will show preliminary data that 
older obese subjects have an accelerated loss of muscle mass during bed rest.  
Mitochondrial dysfunction has been hypothesized to be involved with skeletal muscle insulin 
resistance in older adults with T2D, possibly by affecting IMCL accumulation (71, 90, 91). Our 
group has shown that obesity and T2D is characterized by impaired fatty acid oxidation (17), 
ceramide accumulation (2, 14, 17), and mitochondrial dysfunction (97, 116). These are all factors 
that likely contribute to lower protein synthesis in obese individuals during disuse, resulting in 
greater muscle atrophy and poor recovery. Indeed, recent evidence suggests that the link between 
mitochondria and IR extends to mitochondrial cardiolipin remodeling (68), oxidative stress and 
cellular redox state (5, 67). The contribution of dysfunctional mitochondrial energetics and 
lipotoxicity to muscle atrophy in humans has not been elucidated. Studies in older adults with T2D 
will provide a pathophysiological context and parallax view of mitochondria’s relationship with 
muscle atrophy and recovery that will allow us to delineate novel mechanisms. 
The role of adipose tissue and crosstalk with skeletal muscle. Recent evidence suggests that 
adipose tissue is not just a “storage depot” for fat but is a highly active endocrine organ, which 
plays a pivotal role in whole body energetics (63). In particular, the adipose tissue of older adults 
and T2D becomes infiltrated with macrophage and becomes “inflamed” releasing adipokines 
(TNF) that impact metabolic health of other tissues in the body. Adipose tissue inflammation and 
the release of factors (adipokines) mediate insulin resistance in muscle, which in turn prevents 
protein synthesis and contributes to loss of muscle mass in sarcopenia (61). While these 
mechanisms have been relatively well described in animal models, evidence in humans is lacking. 
An exploratory aim of this protocol will be to examine adipose tissue metabolism/signaling and 
its role in disuse and exercise recovery. 
Effective therapeutic strategies to prevent muscle atrophy and aid in recovery are lacking. 
Pharmacological therapies to date have focused on increasing muscle mass and include 
testosterone, selective androgen receptor modulators (SARMS), growth hormone, and myostatin 
antibodies that are currently at early phase clinical trials (81). Dietary supplements enriched with 
leucine and essential amino acids can promote protein synthesis (119). There is a critical need, 
however, to test mechanism-based approaches to prevent or attenuate atrophy and aid recovery 
following periods of inactivity for older adults. 
Exercise as a strategy to aid recovery. Exercise interventions can clearly improve mitochondrial 
energetics, alleviate IR and stimulate protein synthesis resulting in improved muscle mass and 
strength (10). However, older adults with T2D are reported to have an attenuated improvement in 
health outcomes following exercise (109). In context of recovery from bed rest, the efficacy of 
exercise to facilitate recovery of muscle mass, strength and function in older adults with T2D has 
not been determined. We have shown that aerobic exercise improves insulin sensitivity, 
mitochondrial function and lowers sphingolipid in muscle from older adults and in obesity (33), 
and others have demonstrated reductions in oxidative stress (102). Here we propose to test the 
efficacy of exercise to facilitate recovery following bed rest, and to use exercise as a physiological 
tool to examine the relationships between improved mitochondrial energetics, lipid partitioning, 

 
 
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protein synthesis and IR during recovery. This could also emphasize mitochondria as an adjunct 
target to anabolic therapies. 
 
Study Objectives 
 
The overall objective of this project is to decipher the temporal sequence by which exercise 
training alters mitochondrial dysfunction and muscle protein synthesis in older adults with T2D 
and how an exercise intervention can aid in recovery of muscle mass, strength and physical 
function following bed rest. Studies in the clinically relevant context of aging, T2D, and exercise 
will provide a physiological framework in which the temporal and mechanistic relationships 
between mitochondria, intramyocellular lipid and protein synthesis/breakdown can be interrogated 
during atrophy and recovery. 
 
Study Design 
Research Design 
 
The proposed clinical study is a prospective, parallel two arm, randomized controlled study 
consisting of five discrete but connected phases: 
 
I) Screening, II) Pre-Bed Rest, III) Bed Rest, IV) Ambulatory/Exercise Recovery and V)  
Optional Resistance Exercise  
 
Eligible participants will be Older Adults with T2D and will be randomly assigned to one 
of two groups:  Exercise recovery (EX) or Ambulatory recovery (CON).  
 
The expected duration for a subject in this study could be up to 16 weeks. 
 
A series of physiologic and clinical/health outcomes will be assessed before, during and 
after the bed rest intervention period and recovery period. 
 
Males and females with pre-diabetes or type 2 diabetes in the age range of 60-80 years will 
be recruited from AdventHealth’s electronic medical record (EMR), the TRI recruiting 
database, senior centers, television, radio and social media advertisements, mass mailing 
lists from the Orlando, FL catchment area, and by contacting potential participants on the 
TRI’s participant database. 
 
Study Intervention Description  
 
Bed Rest Intervention. The participant will remain in bed rest for 10 days. The 
metabolically balanced study diet will be individually formulated for each participant based 
on their energy needs and food preferences. Each subject’s protein intake will equal to 
0.8g/kg body weight /d. The remaining macronutrients will be distributed approximately 
to the subject’s typical diet from the 3-day food diary. Required energy intake (kcal/d) will 
be based on RMR results with an activity factor (AF) of 1.1. Diets will be prepared in the 

 
 
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Metabolic Kitchen at the TRI. Participants will maintain strict bedrest and remain flat as 
much as possible during bed rest; however, participants can have the head elevated to 30 
degrees (by study staff) for meals. Participants will be free to change position while lying 
in bed by rolling side to side. Participants may elevate legs when lying flat in bed for 
comfort.  Participants may also sit up during use of incentive spirometer.  Participants may 
be transferred on to a gurney/stretcher, or wheelchair for transportation. Participants will 
be allowed to use a bedside commode for urination and will be taken to a toilet in a 
wheelchair for bowel movements. Participants will be allowed to shower as long as 
subjects use a shower chair or wash up at the sink while sitting in the wheelchair, and/or 
they can take a sponge bath while in bed. Participants may have their hair washed while in 
bed. Participants will not be allowed to take a traditional bath. Participants will be allowed 
to shower normally after becoming ambulatory. This is an intervention that has been shown 
by our group to produce robust reductions in lean mass in older adults (IRBNet #514957-
98) (107). 
 
Exercise Recovery Program. The exercise recovery program will be supervised by a 
certified exercise physiologist and will take place at the TRI exercise training facility. 
Exercise compliance will be monitored carefully. We are examining the muscle-specific 
effects of the recovery exercise program on the muscle groups of interest. We will use an 
exercise recovery program that has demonstrated substantial increases in quadriceps muscle 
size and strength in elderly people (37, 40, 105). During the first 4 weeks of recovery, 
participants will perform a combination of aerobic and resistance exercise training with a 
certified exercise physiologist 3 days per week at the TRI exercise training facility. Exercise 
compliance and adherence will be monitored carefully. Aerobic training will be 30 minutes 
of brisk walking, jogging, and cycle ergometry. Walking will be the primary mode of aerobic 
exercise given its widespread popularity and ease of administration across a broad segment 
of the older adult population. Walking will be at a moderate intensity and determined based 
on ratings of perceived exertion. Three sets of 8–12 repetitions of each resistance exercise 
will be performed to target the major lower body muscle groups, including the vastus 
lateralis of the quadriceps. 
 
Ambulatory Recovery. Participants in the ambulatory recovery group will not receive any 
exercise intervention or advice on exercise. Rather these participants will return to their 
regular daily routine that they engaged in prior to the bed rest intervention. 
 
(Optional) Resistance Exercise Program. The resistance exercise program will be 
supervised by a certified exercise physiologist and will take place at the TRI exercise training 
facility. Exercise compliance will be monitored carefully. Strength training will consist of 
upper and lower body exercises. Participants will participate in strength training 3 days per 
week (12 total sessions). Each resistance exercise will be performed with a resistance that 
will allow 8–10 repetitions (approximately 70-80% of 1 RM). Three sets of 8–12 repetitions 
of each exercise will be performed to target the major upper and lower body muscle groups 
after appropriate rests. Each repetition will employ a slow speed (approximately 5 seconds) 
to maximize muscle hypertrophy and minimize risk of a muscle pull or injury. The subject 

 
 
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will walk at their usual pace before and after strength training to allow for a warm up and 
cool down. Strength training (with an appropriate warm up and cool down) will take 
approximately 1 hour. Speed of contraction for the concentric component will be 
approximately 2 seconds for full extension and the eccentric component will be 4 to 6 
seconds in duration. The one repetition maximum will be determined weekly to ensure that 
each participant is exercising at the appropriate duration. This is the exercise regimen that 
has demonstrated substantial increases in muscle size and strength in elderly people. 
If the participant is unable to come in for a scheduled exercise appointment, alternate 
arrangements for the visit may be made within the visit window with Principal Investigator 
approval. 
 
Study Site(s)/Location(s) and Number of Subjects 
 
 
AdventHealth site locations: Translational Research Institute, 301 E. Princeton Street. 
Orlando, Florida 32804. 
 
Name of external site(s) outside of AdventHealth: N/A 
Estimated number of subjects at external sites:0 
 
Total number of all sites: 1 
Estimated number of subjects at all sites combined: 75 
 
Multi-Site Research Logistics/Communication Plan 
 
N/A 
Research Conducted in a Foreign Country 
 
N/A 
Community-Based Participatory Research 
N/A 
 
Subject Selection 
 
Vulnerable Populations (if applicable) 
 
 
AdventHealth Employees:  Recruitment efforts will follow AdventHealth recruitment 
SOPs for research.   AdventHealth Employees will not be individually targeted nor 
excluded from study participation based on employment.  AdventHealth employees who 
engage the TRI asking to participate in the study will be processed per standard consent 
procedures for participants.  In addition, during the consent process, the study staff will 
review standard consent language stating that an employee’s participation or lack of 
participation in the study will not affect their employment status or relationship with 
AdventHealth.   

 
 
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Inclusion Criteria 
1. Participant must be male or female, ages 60 through 80 years of age. 
2. Participant has pre-diabetes or has been diagnosed with type 2 diabetes and 
taking 0-3 oral hypoglycemic agents, which include basal insulins, incretin 
mimetics, DPP-4 inhibitors, Sulfonylureas and/or Metformin therapy, and has 
an Hgb A1C < 9.0%.   
a. Participants on Insulin, , SGLT2 inhibitors, and Thiazolidinediones will be 
excluded. 
b. Pre-Diabetics are defined as having an HgbA1c of greater than or equal to 5.7% 
and less 6.5%, or a fasting glucose of greater than or equal to 100 mg/dl and less 
than 126 mg/dl or has a glucose of greater than or equal to 140 mg/dl and less 
than 200mg/dl at the 2 hour blood draw during OGTT 
3. Participant must have renal function with an estimated glomerular filtration 
rate (eGFR) > 45 ml/min/1.73m2 determined at screening. 
4. Participant’s triglyceride level is < 350 mg/dl and LDL cholesterol is ≤ 150 
mg/dl at screening. 
5. Participant states willingness to follow protocol as described, the prescribed 
activity level and completing any forms needed throughout the study. 
6. Participant has voluntarily signed and dated an informed consent form, 
approved by an Institutional Review Board/Independent Ethics Committee, 
and provided Health Insurance Portability and Accountability Act 
authorization (HIPAA) or other privacy authorization prior to any 
participation in study. 
 
Exclusion Criteria 
1. Participant has type 1 Diabetes. 
2. BMI > 40.0 kg/m2 
3. Participant is actively pursuing weight loss and/or lifestyle changes. 
4. Participant has a history of pressure ulcers. 
5. Participant has a stated history of Deep Vein Thrombosis (DVT), pulmonary 
embolism, or a known hypercoagulable condition, or other clotting or bleeding 
disorders. 
6. History of gastrointestinal or intracranial hemorrhage. 
7. History of stroke or cerebrovascular accident within the last 6 months. 
8. Recent history of major trauma (within 3 months). 

 
 
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9. Thrombocytopenia (<100,000/microL) or hyperkalemia (K > 5.2) on screening 
laboratory assay.  May repeat lab value per PI discretion.  
10. Untreated or poorly controlled hypertension (SBP > 150, DBP > 95), or hypotension 
(SBP <100 DBP <60) 
11. Participant has hypothyroidism (TSH ≤(0.5mIU/L) or hyperthyroidism TSH ≥ 
10mIU/L. 
12. Participant has current infection (requiring prescription antimicrobial or antiviral 
medication, or hospitalization), or corticosteroid treatment (with the exception of 
inhaled or topical steroids) in the last 3 months prior to screening visit. 
13. Participant is currently taking anti-inflammatory medication or has had anti-
inflammatory medication within 1 week prior to screening (including over the counter 
formulations; e.g. Aleve, Motrin, ibuprofen, naproxen, low dose asprin).   
14. Participant has had surgery requiring > 2 days of hospitalization in the last 1 month 
prior to screening visit. 
15. Participant has an active malignancy or autoimmune disease. 
16. Participant has current significantly impaired liver function in the opinion of the study 
Medical Investigator (mild asymptomatic fatty liver is acceptable), or hepatic enzyme 
tests are ≥ 2.5 times normal limit. 
17. Participant has a chronic, contagious, infectious disease, such as active tuberculosis, 
Hepatitis B or C, or HIV, per self-report. 
18. Participant is an amputee and/or has presence of partial or full artificial limb. 
19. Participant has had a significant cardiovascular event (e.g. myocardial infarction, 
stroke) ≤ 6 months prior to screening visit; or stated history of congestive heart 
failure; or participant has evidence of cardiovascular disease assessed during the ECG 
at screening. In the event of a positive stress test, participants are referred to their 
primary care physician. If the electrocardiogram (ECG) is determined to be a false 
positive, participant may be allowed to participate in study after confirmatory records 
obtained. 
20. Participant currently has uncontrolled severe diarrhea, nausea or vomiting. 
21. Participant has an obstruction of the gastrointestinal tract, inflammatory bowel 
disease, short bowel syndrome or other forms of gastrointestinal disease such as 
stage III or above gastroesophageal reflux disease, gastroparesis, peptic ulcer 
disease, celiac disease, intestinal dysmotility, diverticulitis, ischemic colitis and 
bariatric surgery. 
22. Participant cannot abstain from alcohol for the pre-bed rest and bed rest portion of the 
study. For the recovery portion of the study the participant must agree to consume no 
more than ~14gm of alcohol per day (equivalent to ~ 1 glass of wine (4-5 oz 12% 
ABV) or ~bottle of beer (12 oz 5% ABV) /day). 

 
 
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23. Participant cannot refrain from taking medications/dietary supplements/herbals or 
substances that could modulate glucose metabolism (other than oral hypoglycemic 
medications), or are considered anabolic, or reduce weight (fat mass), or that may 
interact with low-molecular weight heparin or induce hypo- or hyper-coagulable 
state, in the opinion of the PI or medical investigator, starting one week prior to Pre-
bed rest phase and over the entire course of the study. These include progestational 
agents (except prescribed birth control), steroids, growth hormone, dronabinol, 
marijuana, calcium-beta-hydroxy-betamethylbutyrate (CaHMB), free amino acid 
supplements and dietary supplements to aid weight loss. 
24. Participant has a mini-Mental State Examination score < 21. 
25. Subjects who fulfill any of the contraindications for MRI; examples include metal 
implants, devices, paramagnetic objects contained within the body and excessive or 
metal-containing tattoos. 
26. Unable to participate in MR or DEXA assessments due to physical limitations of 
equipment tolerances (e.g., MRI bore size and DEXA 450-pound weight limit), 
claustrophobia, or based on Investigator’s judgment at screening. 
27. Participant has a sensitivity or allergy to lidocaine. 
28. Participant has a sensitivity or allergy to heparin, enoxaparin, or other low molecular 
weight heparin. 
29. History of allergy to pork products. 
30. History of heparin-induced thrombocytopenia. 
31. Hemoglobin < 10.0 g/dL for females; < 11.0 g/dL for males; or participant has 
clinically significant signs/symptoms of anemia in the opinion of the PI or medical 
provider. 
32. Concomitant medications with known contraindication or interaction with low-
molecular weight heparin (including anti-platelet agents, anti-coagulant agents, non-
steroidal anti-inflammatory drugs) 
33. Presence of any condition that, in the opinion of the Investigator, compromises 
participant safety or data integrity or the participant’s ability to complete the study. 
34. Participant experiences symptoms of claudication. Symptoms include cramping pain 
in the legs and/or difficulty walking.    
 
Because all women participating in this project will be post-menopausal, there will be no need 
for a pregnancy test prior to DEXA procedures. Females currently on hormone replacement 
therapy (HRT) can participate in the study if they have been on a stable dose of HRT for at least 
6 months and will continue to be on HRT during the study. 
 
Potential participants taking stable doses of medications for the last 30 days prior to screening 
for Blood pressure, cholesterol, GERD may be permitted to participate. 

 
 
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Resources Available 
We attest that all TRI faculty and staff will be trained, and this training will be documented.  
We will adhere to AdventHealth ORA SOP 06 (Research Personnel Selection, 
Qualification, Responsibilities, and Training) and Working Instruction (WI) 031.100.015 
Documentation of Protocol Training. 
 
Protocol development meeting, with follow-up meetings, will occur regularly to ensure that all 
persons assisting in the research are adequately informed. Periodic meetings will be scheduled to 
maintain and update any new information/issues. 
 
Study Procedures 
 
Subject Recruitment and Screening 
 
Males and females with pre-diabetes and type 2 diabetes in the age range of 60-80 years 
will be recruited from AdventHealth EMR, senior centers, television, radio, and social 
media advertisements, mass mailing lists from the Orlando, FL catchment area, and by 
contacting potential participants on the TRI’s participant database.  
 
Potential participants will be medically screened to determine good health, weight stable 
(no gain/loss of ≥ 10 lbs in 6 months prior to screening), and without any contraindication 
to exercise. Approximately equal numbers of men and women will be recruited. Neither 
race nor ethnicity will be exclusions. Re-screening of participants may be repeated per 
Medical Investigator discretion, if needed. 
 
We will recruit volunteers through public advertisement by placing advertisements in 
community newspapers and radio, as well as on social media. Individuals who respond to 
the ads will be contacted by telephone for preliminary screening and will be given an 
opportunity to ask initial questions and be provided additional description of the study. 
Potential volunteers who seem to meet criteria will be scheduled for an outpatient 
examination at TRI. Informed, written consent will be obtained at the screening visit.  
 
We anticipate ~400 phone screens to successfully complete 18 participants per group.  
Consent Process  
We attest that all study staff delegated the authority to obtain informed consent 
will follow The institutional “SOP:  401.116 Informed Consent Process and 
Written Documentation of Informed Consent “and Investigator Guidance: Remote 
Informed Consent Process in Non-Exempt Research (HRP-831)” and Investigator 
Guidance: Remote Informed Consent Process in Non-Exempt Research (HRP-
831)”. 

 
 
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Potential subjects will be given time to read the consent document and will be 
interviewed by a clinical coordinator. The coordinator will give a detailed 
overview of the study and then ask if the potential subject has questions and if they 
might be interested.  The candidate is then encouraged to ask questions. After all 
questions have been addressed and interest remains, the candidate then signs the 
consent if he/she chooses to do so. After the subject has signed the consent or in 
the scenario of the remote Inform Consent process once the original signed copy 
has been received, inclusion and exclusion criteria will be reviewed. The new 
subject is given a photocopy of the signed consent form for his/her records and the 
PI/Sub-I will review all participant Inclusion Exclusion Worksheets.  Once 
deemed eligible, the PI/Sub-I will sign and date the Inclusion Exclusion 
Worksheet.  Participants will be free to withdraw at any time for any reason 
without consequence. 
All study staff delegated the authority to obtain informed consent will follow 
SOP:  401.116 Informed Consent Process and Written Documentation of Informed Consent. 
Non-English Speaking Subjects 
The participant population that we recruit will be primarily English speaking.  In 
the unlikely event that a non-English speaking participant meets criteria for 
enrollment, the AdventHealth IRB Investigator Guidance: Short-Form Consent 
Process in Research (HRP-804), will be followed. 
Subjects who are not yet adults (infants, children, teenagers) 
 
 
We are not enrolling participants who are not yet adults. 
 
Cognitively Impaired Adults 
 
 
We will not enroll cognitively impaired adults. 
 
Adults Unable to Consent 
 
 
We will not enroll adults who are unable to provide consent. 
 
Documentation of Informed Consent Process 
 
Documentation of the informed consent process is required to establish that the subject was 
accurately and adequately informed and that no study-related procedures were initiated 
prior to obtaining informed consent.  A research team member will note in the source 
documentation the consent process, date consent was obtained, and that consent was 
obtained prior to initiating any research procedures. In the scenario of Remote Informed 
Consent Process documentation of all actions will be maintained as described in the 

 
 
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Investigator Guidance: Remote Inform Consent Process in Non-Except Research (HRP-
831).   
 
 
Waiver of Written Documentation of Consent or Waiver of Consent  
 
N/A 
 
Randomization 
 
A randomization schedule containing the subject treatment group assignment (EX or CON) 
will be generated by the study statistician. A permuted blocks approach will be used with 
subjects stratified by gender. We will use blocks of random sizes of 4 to reach the total 
sample size of 20 to start (18 completers) in each group. 
 
Study Visits  
 
All study visits will be conducted at the AdventHealth Translational Research Institute.  
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1: Schematic depiction of the study protocol.  
 
 
Phase 
Description 
Duration 
Subject at home 
Subject Ambulatory 
Visit Window 
I 
Screening 
 < 35 days 
Yes 
Yes 
35 days within 
PBR1    
II 
Pre-Bed Rest 
5 days  
Yes 
Yes 
- 
III 
Bed Rest 
10 days  
No 
No 
- 
IV 
Ambulatory/Exercise 
Recovery 
 4 weeks 
Yes 
Yes 
+ 1 days 
V 
Optional Resistance 
Exercise  
 4 weeks 
Yes 
Yes 
+ 2 days 
 

 
 
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Phase I – Screening. 
 
Screening Visit 1  
Approximate visit time:  ~ 4 hours 
 
The purpose of the staged screening process is to identify and verify eligible participants 
over a series of contacts. Interested participants are first screened by phone. The phone 
interview is designed to exclude individuals who are clearly ineligible or unlikely to benefit 
from participation in the study. Potentially eligible volunteers will arrive at TRI in the 
morning in a fasted state (at lease or ~8-hour overnight fast). Following provision of 
voluntary written informed consent, the following tests will be conducted. The study menu 
will be reviewed with each subject at screening and reasonable substitutions will be made 
for diet intake compliance.   
 
 
Assessment 
Screening Visit (days) 
1 
2 
Review and collect signed ICF 
X 
  
Review Eligibility (may occur remotely) 
X 
 
Health history(may occur remotely) and full physical 
examination1 
X 
  
Medication/supplement history (may occur remotely)  
X 
  
Age 
X 
  
Metabolic Body Weight16 
X 
 X 
Height 
X 
  
Vital Signs11 
X 
  
BMI 
X 
  
Waist circumference4 
X 
 
Questionnaires (CHAMPS, Mini-Mental, etc.)12 
X 
  
SPPB7 
X 
  
Step Test15 
X 
 
400m walk test (usual pace)13 
X 
 
Handgrip strength10 
X 
  
Fasted blood draw2 
X 
  
OGTT (Pre-Diabetics only) 
X 
 
Resting 12 lead ECG5 
X 
  
Urine Collection3 
X 
 X 
Dispense 3-day food diary 
X 
 
Collect 3-day food diary 
 
X 

 
 
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Placement of Accelerometers 
  
X 
Resting Metabolic Rate/RQ9 
 
X 
VO2max test/stress test8 
  
X 
 
1A Health History will be obtained. A standard full physical examination will be performed 
by a study physician, physician assistant, or nurse practitioner. Age, weight, height and 
BMI and hand grip strength will also be determined. 
 
Medical Health History and Physical will be conducted by a study physician or medical 
provider (nurse practitioner or physician assistant) for enrollment into the study. 
 
2A fasting blood draw for determination of lipid profiles (cholesterol, HDL, LDL, VLDL, 
triglycerides); Complete Metabolic Panel (CMP) which includes electrolytes, glucose, 
kidney and liver function (ALT, AST, Alk phos); Complete blood count with platelet and 
differential (CBC); kidney function (BUN and creatinine); HbA1c, Thyroxine (T4), 
Thyroid stimulating hormone (TSH). Total blood volume for screen labs is approximately 
18mls. Safety labs may be repeated per Medical Investigator discretion, if needed. 
 
3Urine Collection for urinalysis will be collected during SV1. Urine collection for urea 
nitrogen and creatinine will be completed during SV2. 
 
4Waist Circumference Measurements which are a surrogate measure of abdominal fat will 
be conducted. The Gulick II tape measure will be used for accuracy in obtaining duplicate 
waist measurements. All circumferences will be measured in centimeters directly on the 
skin. 
 
5Resting 12 lead ECG: Electrodes will be placed on the chest to provide a tracing of the 
electrical activity of the heart. 
 
Oral Glucose Tolerance Test (OGTT): Glucose tolerance will be assessed only in 
potential pre-diabetics with a 75 g oral glucose tolerance test (OGTT).  Subjects will be 
studied after an overnight fast.  Samples may be collected from an indwelling IV line or 
by venipuncture.  (Consider the necessity of multiple time points/analytes for the given 
experiment) Two baseline samples will be drawn at approximately times= -10 and -1 
minutes.  The subjects will then ingest a 75 g glucose beverage at time=0 minutes (first 
sip of beverage) and will be asked to consume the entire beverage in <5 minutes.  Blood 
samples will be collected at approximately times= 30, 60, and 120 minutes.  Each blood 
sample will be analyzed for plasma or serum glucose, insulin, free fatty acid, C-peptide, 
and incretins (GLP-1 and GIP).  Total blood volume collected during this procedure is 
approximately 48 mL. 
a. Participants will be considered pre-diabetic if their HgbA1c is greater than or 
equal to 5.7% and less 6.5%, or a fasting glucose of greater than or equal to 100 

 
 
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mg/dl and less than 126 mg/dl or has a glucose of greater than or equal to 140 
mg/dl and less than 200mg/dl at the 2 hour blood draw during OGTT 
 
7Short Physical Performance Battery (SPPB) 
Briefly, this test measures standing balance (feet side by side, semi-tandem, and tandem), 
habitual gait speed (4-meter walk test), and chair stand time (how rapidly an individual can 
stand from a seated position). Additionally, each participant will be asked to complete a 
timed step test. 
• Standing Balance should be performed on a flat non-slip surface and without 
assistance, such as a cane or walker. 
• The Four Hundred Meter Walk Test (400MWT) is a practical, simple test that 
requires an obstacle free hallway. This test measures the walking speed in meters 
per second over 4m. For the 4MWT, clearly mark the 4 m course using high 
visibility, or similar tape. 
• For the Chair Stand test the subject is seated, with their arms crossed. The subject 
must be able to stand upright and sit back down from this seated position. 
 
13A 400-meter walk test at usual pace will also be conducted. The 400-meter walk test is a 
measure of cardiorespiratory fitness, lower extremity muscle function and general 
mobility. During this self-paced walking test, patients are instructed to walk 400 meters at 
their usual pace or without any expectation of time. The ability to walk 400 meters in less 
than 15 minutes has been suggested as an indicator of sufficient capacity for community 
ambulation. The goal of this outcome measure is to determine the amount of time required 
to walk 400 meters. The patient may rest whenever necessary but should understand the 
aim is to walk the entire 400-meter distance as soon as possible. The time needed to 
complete the test will be recorded on source documentation. If the person is unable to walk 
the full 400 meters because of needing to stop or not completing the distance within 20 
minutes, the distance (in meters) and time walked during the test will be recorded.  
 
The 400mwt should be performed using a 20 m course. The 400-meter walk test will be 
performed and timed using a stopwatch. Subjects will be instructed to walk as quickly as 
possible for 10 laps around markers placed 20 meters apart.  Standard encouragement will 
be given by study personnel after the completion of each lap.  
 
10Subjects will perform a hand grip test that will be conducted three times with 1 minute 
of rest between each repetition using the dominant arm. The greatest grip strength and the 
average of the three tests will be recorded.   
 
11Vital signs consist of body temperature, pulse rate (or heart rate), blood pressure, and 
respiratory rate. 
 
12A number of health questionnaires will be administered at screening, including: 
CHAMPS Physical activity, Life Space, Lifestyle, Functional Status, Pittsburgh 
Fatigability Scale. 

 
 
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15The Step Test is conducted by recording the time it takes for a participant to complete a 
series of repeated steps.  The participant will be asked to complete a timed step test where 
they will be repeatedly stepping up and down from an 8-inch high step.  
 
16 Body Weight is Metabolic Weight. Metabolic weight is completed during fasting and 
with an empty bladder while wearing a gown and underwear. An average weight between 
two measurements will be used. The metabolic weight will be the difference between of 
the average subject weight and weighted gown.  
 
Screening Visit 2  
Approximate visit time:  ~ 2 1/2 hours 
 
9Resting Metabolic Rate/Respiratory Quotient (RMR/RQ): Indirect calorimetry will 
measure the resting metabolic rate/respiratory quotient (RMR/RQ) and substrate utilization 
after an 8-hour overnight fast using a MAX II Metabolic cart (AEI Technologies, 
Pittsburgh, PA).  The analyzer will be calibrated before each subject with standardized 
gases containing 5% CO2, 21% O2 and balance N2. Subjects will be instructed to lie in a 
Semi-Fowler’s position and remain motionless and awake during these periods.  A 
transparent plastic hood connected to the device will be placed over the head of the subject. 
Calculations of O2 consumption (VO2) and CO2 production (VCO2) will be made by 
comparing a single measure of the O2 and CO2 concentrations of the testing room to 
continuous measurements of O2 and CO2 concentrations in expired air diluted in a constant 
air flow (20-50 L/min) generated by a pump.  From the above, energy expenditure 
standardized for temperature, pressure, and moisture will be calculated at one-minute 
intervals.    
 
8Graded exercise testing: Volunteers must pass the medical screen before they will be 
allowed to undergo the exercise test. If the resting ECG prior to the exercise test shows 
clinically significant abnormalities, the test will not be conducted. If abnormalities develop 
during the test, the test will be stopped. On both occasions, the participant will be referred 
to their Primary Care Physician for further evaluation. Before and after exercise activities 
are performed, the participant’s that are type 2 diabetics will have a blood glucose  checked 
using their home glucose monitor or glucose monitor at TRI. Blood glucose will be 
measured prior to and immediately following each exercise session for safety. If blood 
glucose is > 300mg/dl, study team will notify TRI provider immediately for review of 
glucose values, as well as medications.  If the glucose values are consistently > 400mg/dl, 
participant will be instructed to follow up with their primary care physician. If blood 
glucose is < 100mg/dl, a snack will be given, and the participant will be allowed to exercise. 
Self-monitored blood glucose measurements should be checked anytime a patient has 
symptoms of hypoglycemia or hyperglycemia.   
 
Aerobic fitness will be determined by measuring maximal O2 consumption (VO2max) 
during a stationary bicycle exercise test.  Provider monitoring will follow The American 

 
 
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College of Sports Medicine’s (ACSM) Guidelines for Exercise Testing and Prescription. 
Heart rate, blood pressure and ECG, if applicable, will be recorded throughout this test. 
During the test, subjects will breathe through a low resistance mouthpiece and wear a nose 
clip. Expired gases will be measured by indirect calorimetry and heart rate monitored by a 
portable heart rate monitor and/or ECG.   
 
Following a standardized warm up, subjects will begin exercising at a moderate intensity 
with the workload (resistance on a cycle ergometer or speed and grade on a treadmill) 
increased every minute until the subject can no longer continue. VO2 max tests will take 
as little time as possible in order to ensure that test termination is due to the subject reaching 
VO2 max rather than terminating the test due to muscle exhaustion. A leveling-off (plateau) 
or decline in oxygen uptake should be demonstrated in order to be reasonably sure that a 
subject has achieved the maximum capacity for aerobic metabolism. However, there are 
several additional criteria that will be used to determine if maximal aerobic capacity is 
achieved. These criteria are: the respiratory exchange ratio (RER) increased to 1.10 or 
higher and the subject’s heart rate increased to within 10 beats of the age-predicted 
maximum (208 – [0.7 x age]).   
 
The test will also be stopped, and subjects excluded from further participation if there is 
>2mm ST-segment depression on ECG or evidence of cardiovascular instability, such as 
hypotension, dangerous arrhythmias or angina.  
 
Prior to beginning the exercise test, the Borg Rating of Perceived Exertion (RPE) Scale 
will be presented to participants. The RPE Scale is a rating of how hard a person feels their 
body is working and is an accepted supplementary tool for prescribing exercise programs 
in both healthy and special populations. Throughout the test, participants will be asked to 
point to the numerical rating of how they feel and will be encouraged to remember a rating 
of how “somewhat hard” (13) feels. The rating of “somewhat hard” should coincide with 
a heart rate of approximately 50-70% of their maximal exertion level. During maximal 
testing participants should report a rating of 19-20 (“maximal exertion”). A cardiologist 
will review the ECG, if the medical Provider deems necessary. If a study physician or 
cardiologist identifies any artifacts observed from the exercise test that may render the 
participant ineligible for participation in an exercise intervention, that subject will be 
referred to his/her Primary Care Physician (PCP) or cardiologist for follow-up care and 
further examination of test results. If participants are referred for further examination, a 
written approval to perform unsupervised exercise must be obtained by PCP or cardiologist 
in order for subject to continue participation in the research study. If written approval from 
the PCP or cardiologist is not able to be obtained, participant will be excluded from further 
participation in the investigation. The test will take approximately 30-40 minutes. 
 
Accelerometers to assess free-living physical activity over approximately 5 days will be 
provided, along with instructions on how to use the devices. They will be placed on the 
arm and wrist. The device will be returned by the participant after at least 5 days of 
recording and prior to Pre-Bed Rest Day 1. 

 
 
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Phase II – Pre-Bed Rest (PBR). 
This phase of the study should be initiated no more than 35 days after the first screening 
visit. PBR 1 will be considered the study baseline. The Table below summarizes the 
schedule of assessment during Pre-Bed Rest. 
 
The five days prior to bed rest is considered the pre bed rest period and will be abbreviated 
as PBR 1 to PBR 5. Subjects will be required to report to the study site for meals and 
assessments during these 5 days. To prepare subjects for the bed rest diet (stabilize diet), 
subjects will be provided with a breakfast, lunch and dinner, for consumption at the study 
site or as packaged meals for each of the 5 days. Subjects will consume breakfast at the 
study site and will have the option to consume lunch and dinner at the study site, or they 
will be provided with a packed lunch and dinner for later consumption. 
 
The metabolically balanced study diet will be formulated for each subject to have each 
subject’s protein intake equal to 0.8g/kg body weight/d, and kcal intake to be based on their 
measured total daily energy expenditure from the armband accelerometer. The remaining 
macronutrients will be distributed approximately to the subject’s typical diet from the 3-
day food diary. 
 
Body Weight during the Bedrest phase only, will be taken using the bed scale during fasting 
and with an empty bladder while wearing a gown or light clothing and undergarment(s). 
 
 
 
 
 
 
*The imaging and exercise procedures may be performed as baseline measurements 
on any Pre-Bed Rest day  
 
1MR Assessments to occur preferentially on PBR2, however may occur on any PBR 
day to accommodate scheduling.  Assessments defined below: 
Maximal ATP Production (ATPmax): ATPmax will be measured in order to determine in 
vivo mitochondrial capacity. Participants will complete the MR Screening for ATPmax, and 
Assessment 
Pre-Bed Rest (days) 
1 
2 
3 
4 
5 
Medication changes 
 X 
X  
X 
X  
X 
Metabolic Body Weight16 
X  
X  
X  
 X 
X 
Evaluate DVT Risk 
 
 
 
 
X 
MR Assessments1 (ATPmax//IMCL/IMAT/) 
  
X* 
  
  
  
Biodex/Strength assessment2 
  
  
 X* 
  
  
DEXA3 
 
 
X* 
 
 
Eucaloric Diet Stabilization - Adequate 
protein 
X 
X 
X 
X 
X 
Admit patient for bed rest intervention 
  
  
  
  
X 

 
 
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the information will be verified with the participant by a member of the TRI Imaging Core 
to determine if data acquisition can be performed safely.  The magnetic resonance scans to 
measure mitochondrial energetics will be performed on a 3T Philips Acheiva at the TRI.  
The participant will be informed that the imaging scans will produce loud “knocking” 
noises, but they will be provided both ear plugs and earphones to minimize this noise.   
OVERVIEW:  The Phosphocreatine (PCr) recovery time constant (τ) and the PCr level in 
oxygenated muscle at rest (PCr)rest will be used to calculate maximum mitochondrial 
capacity (ATPmax).  Participants will be asked to lie supine on the patient table of the MRI 
for approximately up to one hour.  After set of survey images is acquired to locate the 
muscle group of interest, a fully relaxed 31P spectra will be collected. The ATPmax 
experiment will be performed by obtaining a set of sequential 31P spectra; one spectrum 
will be acquired every 6-7.5 seconds for the duration of the ATPmax experiment.  For the 
first two minutes, the participant will remain still in order to obtain baseline [PCr], [ATP] 
and [Pi] measurements.  After the baseline is established, the participant will be asked to 
perform contractions of the quadriceps (by kicking against straps around the lower leg) for 
up to 45 seconds. After kicking is stopped, the participant will remain still for the remainder 
of the experiment in order to allow the PCr peak to return to baseline. The ATPmax 
experiment may be repeated. The scan time for the ATPmax experiment will take 
approximately 45 minutes. 
 
Intramyocellular Lipid In Vivo 1H Magnetic Resonance Spectroscopy (IMCL):  The 
participant will be asked to lie supine on the patient table of the magnet (Philips Acheiva 
3T) for approximately 45 min. The participant’s leg will be positioned inside a radio 
frequent (RF) 1H knee coil with the knee in extension and the ankle in a neutral position. 
Standard clinical MR Imaging, including 3-place localization and T2 weighted images, will 
be completed to obtain anatomical images for voxel / slice localization. The PRESS (point 
resolved spectroscopy) sequence with water suppression will be used to obtain 
spectroscopic measurements of the intramyocellular lipid (IMCL). A PRESS single voxel 
sequence will be used to obtain spectra from separate voxels (up to 15 x 15 x 15 mm3) 
within largest volume of the calf muscles (echo time (TE) = 35 msec and a resonance time 
(TR) = 1500 seconds). Voxels will be positioned to avoid fascia, vascular structures and 
gross marbling as determined using the anatomical images. 
 
MR assessment of Intramuscular and Subcutaneous Fat (IMAT):  Volumetric 
measurement of fat, muscle and bone in the participant’s mid-thigh leg will be completed 
using an Achieva 3T MRI/Multinuclear MRS (Philips, Andover MA).  Scans will be 
performed under standardized conditions with subjects in a supine position.  The torso XL 
coil will be placed around their legs (encompassing both knees and thighs).  If the 
participant and the coil cannot fit into the bore of the magnet, the coil will be removed and 
the exam will be completed using the quadrature body coil.  Each scan will take 
approximately 30 minutes to perform.  Low resolution scans will be taken to determine the 
approximate midpoint of the participant’s femur.  Then T1 weighted imaging sequences 
will be performed centered at the midpoint.  
 

 
 
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2Strength assessment. The initial weights for knee extension should be set based on the 
subject’s predicted 1 repetition maximum (1 RM). Based on previous research, males and 
females < 65 years of age have a predicted 1 RM of 90% and 60% of their body weight, 
respectively, for the knee extension. For each additional 10 years of age the 1 RM should 
decrease 10% (ex. 91-year-old male would have a predicted 1 RM of 60% of his body 
weight). This number should be calculated and recorded on the data collection sheet. One 
can then calculate the subject’s first 4 lifts at 20, 35, 50, and 70 percent of predicted 1 RM. 
This weight scale is followed, and weight is continually increased until termination of the 
test. If, prior to exercise, the blood pressure is less than 160/100 mm Hg, then the subject 
will warm-up on a bike for ~ 1 minute with no resistance. If BP is elevated the subject 
should rest for 3 more minutes. If BP is still elevated greater than 160/100 mm Hg, the 
subject will be sent home with the recommendation to contact their physician, and this 
subject will be followed up for potential re-testing.  
 
Termination criteria for maximal strength testing: 
- A subject asks to discontinue testing. 
- An individual moves a weight through a full range of motion with relative comfort but 
cannot move through the full ROM with a two-unit increase. 
- Subject cannot move through full ROM with proper form (subject lifts lower or upper 
back away from padded seat, subject kicks pad using acceleration force to lift weight, hands 
not crossed across chest, uneven or jerky ROM). 
 
Relative termination criteria: 
- Subject reports >3 on P/D scale with explanation that the pain persists after the cessation 
of movement or if the subject reports >3 in any area besides the knee, upper or lower leg. 
- Subject reports >18 on RPE scale. 
 
2Biodex Testing. Muscle power testing will be performed using a Biodex pneumatic-
driven dynamometer which is equipped with load cells and potentiometers (for helping 
measure changes in joint angle). The dynamometer is connected to a PC with software to 
measure muscle power and velocity. If the BP is in acceptable range prior to exercise, the 
subject will warm up with one minute of free pedaling on a cycle ergometer (seat height is 
adjusted by setting the seat height at standing hip height and adjusting while subject is on 
the bike for 10o of flexion at the knee when leg is fully extended). Tester explains the power 
test and also describes the responses to the pain/discomfort (P/D) scale (range = 0 to 6) and 
that the subject may decide to discontinue testing at any time. The technician will then 
explain the test: “This is a power test and we want to see how quickly you can move an 
amount of weight through a distance.” After the 1-minute bike warm-up the subject will be 
seated on the Biodex machine with the lateral condyle of the knee lined up with the axis of 
rotation of the machine arm and the seat belt should then be fastened snugly. Both legs will 
be tested, and the first leg will be selected randomly. Familiarization of the Biodex will be 
performed at the first day of testing.  Familiarization at future visits is not required. The 
subject will be instructed to keep arms crossed across the chest and to remember to 
maintain normal breathing patterns. Next the subject performs a power test with 60, 120 

 
 
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and 180 degrees on each leg and gives a rating of P/D. Subject will perform three power 
tests on each leg and a 2 min rest between each increase in resistance. The subject will give 
a P/D rating after each trial. Before each test the tester will remind the subject to keep their 
back against the seat. If necessary other constant reminders will include – “Breathe 
normally,” “Look straight ahead,” or “Try to move your leg up to here (tester indicates end 
of movement).” The tester will also provide standard encouragement such as – “Move your 
leg as quickly as you can,” “Give me your best effort”. After both legs have been tested 
BP will be taken at conclusion of test and after 5-10 minutes of rest. If BP reading is normal, 
testing will continue. If it is above 160/100 mm Hg or has decreased by more than 15 mm 
Hg (top or bottom number) from the resting values, the subject will rest for 2 minutes and 
BP will be taken again. If BP has not normalized to below 160/100 mm Hg, further testing 
will be discontinued, and the subject will remain in the facility for at least ten more minutes 
while monitoring blood pressure. When BP finally normalizes, the subject will be sent 
home with the recommendation to contact their physician. The PI, Dr. Coen, will also be 
notified.  
 
Termination criteria for maximal power testing: 
- A subject asks to discontinue testing. 
- Equipment failure 
Relative termination criteria: 
- Subject reports >3 on the P/D scale (with explanation that the pain persists after the 
cessation of movement or if the subject reports >3 in any area besides the knee, upper or 
lower leg. 
 
Before and after exercise activities are performed, the participant’s blood glucose will be 
checked (type 2 diabetics only) using their home glucose monitor or glucose monitor at 
TRI. Blood glucose will be measured prior to and immediately following each exercise 
session for safety.  If blood glucose is > 300mg/dl, study team will notify TRI provider 
immediately for review of glucose values, as well as medications.  If the glucose values 
are consistently > 400mg/dl, participant will be instructed to follow up with their primary 
care physician. If blood glucose is < 100mg/dl, a snack will be given, and the participant 
will be allowed to exercise. Self-monitored blood glucose measurements should be 
checked anytime a patient has symptoms of hypoglycemia or hyperglycemia.   
 
3Dual Energy X-Ray Absorptiometry (DEXA). DEXA Scans will be performed to 
measure body fat and estimate muscle mass using a GE Lunar iDEXA whole-body scanner.  
The participant will remove all metal accessories and may be asked to change into a 
hospital gown.  The participant will lie on the DEXA table while the scanner arm emits 
low energy X-rays as it passes along the body.  One scan takes up to 15 minutes and the 
radiation dose is less than 1 mrem, less than half the average daily radiation dose in 
America   A urine pregnancy test will be completed on all women of childbearing potential 
(all women except those with prior hysterectomy, tubal ligation, or absence of menses for 
>2 years) prior to the DEXA scan for safety.  DEXA scan will occur preferentially on 
PBR3, however may occur on any PBR day to accommodate scheduling.   

 
 
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Phase III –Bed Rest. 
This phase of the study may be initiated either the evening of PBR 5 or during BR 1. The 
subject should remain on bed rest for approximately 10 days. A metabolically balanced 
study diet will be formulated for each subject to have each subject’s protein intake equal 
to 0.8g/kg body weight/d.  The remaining macronutrients will be distributed approximately 
to the subject’s typical diet from the 3-day food diary. Required energy intake (kcal/d) will 
be based on RMR results with an activity factor (AF) of 1.1. 
 
It is recommended that subjects maintain strict bed rest and remain flat as much as possible 
during bed rest, however subjects can be propped up (by study staff) with pillows (for 
eating, etc.). Subjects are free to change position while lying in bed by rolling side to side. 
Subject may be transferred onto a gurney/stretcher, or wheelchair for transportation. 
Subjects will be allowed to use a bedside commode for urination and will be taken to a 
toilet in a wheelchair for bowel movements. Subjects will be allowed to shower as long as 
they are sitting and wash up at the sink while sitting in the wheelchair, and/or they can take 
a sponge bath while in bed Subjects may have their hair washed while in bed. Subjects will 
not be allowed to take a traditional bath. Subjects will be allowed to shower normally after 
becoming ambulatory. Body Weight during the Bedrest phase only, will be taken using the 
bed scale during fasting and with an empty bladder while wearing a gown or light clothing 
and undergarment(s). 
 
The following procedures, evaluations and/or assessments will be obtained daily during 
bed rest: 1) Medication/supplementation changes, 2) Vital signs consisting of body 
temperature, pulse rate (or heart rate), blood pressure, and respiratory rate will be 
monitored routinely (3 times daily). 3) Subjects will be interviewed to determine if any 
AEs/SAEs occurred in the last 24 hours. Medication to help mitigate reflux problems, 
constipation, and other issues associated with being supine will be available and 
administered per site standard of care or study physician discretion. All medications 
administered will be recorded. 
 
Following 10 days of bed rest, the post bed rest recovery will consist of assisting the 
participant with ambulation and ensuring the participant is capable of showering, walking 
and dressing without assistance. The participant may stay an extra night at the study site 
for further recovery if it is deemed medically necessary by the study physician. A follow 
up phone call will be made that evening to ensure the subject is ambulatory and to inquire 
about any adverse events. The participant will return to the TRI on day 11 (+ 1 day) 
following discharge for additional post-bedrest assessments as listed below. 
 
Before and after exercise activities are performed, the participant’s blood glucose will be 
checked (type 2 diabetics only) using their home glucose monitor or glucose monitor at 
TRI. Blood glucose will be measured prior to and immediately following each exercise 
session for safety.  If blood glucose is > 300mg/dl, study team will notify TRI provider 
immediately for review of glucose values, as well as medications.  If the glucose values 

 
 
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are consistently > 400mg/dl, participant will be instructed to follow up with their primary 
care physician. If blood glucose is < 100mg/dl, a snack will be given, and the participant 
will be allowed to exercise. Self-monitored blood glucose measurements should be 
checked anytime a patient has symptoms of hypoglycemia or hyperglycemia.   
 
Capillary blood glucose will be measured four times a day for type 2 diabetics, before each 
meal and at bedtime. If two consecutive results of > 240mg/dl are recorded, the study 
physician will review and change the patient’s current treatment regimen (DPP-4 
inhibitors, sulfonylureas and/or Metformin) to prevent further hyperglycemia. If two 
consecutive results of >270mg/dl are recorded, the study physician may initiate insulin 
therapy. Insulin therapy will continue until the participant reaches a target glucose range 
of 140–180 mg/dL (7.8–10.0 mmol/L). The participant’s treatment regimen and diet will 
be reviewed and changed as necessary to prevent hypoglycemia (<70 mg/dL), although 
this is not expected with medications like, DPP-4 inhibitors and/or Metformin. 
 
 
The following table summarizes the schedule of assessment during Bed Rest. 
 
Assessment 
Bed Rest (Days) 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
(post 
BR) 
Placement of Accelerometers 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Adverse events 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medication/supplement history 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Eucaloric Diet - Adequate protein 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Vital signs 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Passive Exercise4 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Incentive spirometer5 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Evaluate DVT risk 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Body Weight 
X 
 
X 
 
X 
 
X 
 
X 
X 
Metabolic Body Weight16 
 
 
 
 
 
 
 
 
 
 
X 
Glucose Clamp - 4Hr6 
X 
RMR10 
 
 
 
 
 
 
 
 
 
X 
 
24-Hour Urine 
 
 
 
 
 
 
 
 
X 
 
Blood draw (hemoglobin and 
hematocrit)7 
 
 
 
 
 
 
X 
 
 
X 
 
Deuterated H2O administration8 
X 
X 
Fasting Muscle Biopsy/Blood 
draw1  
X 
Fasting Adipose Biopsy2 (optional) 
X 
 
 
 
 
 
 
 
 
X 
 
Anticoagulant Therapy  
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 

 
 
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DEXA3 
 
 
 
 
 
 
 
X 
MR Assessments 
(ATPmax/IMCL/IMAT3) 
 
 
 
 
 
 
 
X 
Orthostatic Hypotension 
Assessment11 
 
 
 
 
 
 
 
 
 
X 
 
VO2max test 
X 
400m walk test 
 
 
 
 
 
 
 
 
 
 
X 
Biodex/Strength testing 
X 
SPPB 
X 
Step Test 
 
 
 
 
 
 
 
 
 
X 
 
Questionnaires9 
 
 
 
 
 
 
 
 
 
 
X 
 
1Muscle Tissue Biopsy:  For this study fasted is defined as consuming no food (except for 
water and allowed medications/dietary supplements) for at least 8 hours the before blood 
draw/muscle biopsy. Fasting status should be verified verbally with the subject. A 
venipuncture blood draw will also be taken prior to each muscle biopsy (Approximately 
44ml on Day 10)). Blood will be processed, aliquoted and stored at -80C. 
 
Our group has performed more than 1,500 muscle biopsies during the past 20 years (600 
during the past 4 years since the move to the TRI), including same day serial biopsy studies 
and studies in men and women over 90 years old (14, 17-19, 34, 35). We will conduct 1 
biopsy immediately post bed rest (day 10) and 4 biopsies of the vastus lateralis during the 
ambulatory or exercise recovery interventions. The approximate locations of the biopsies 
will be balanced as indicated to avoid interference from previous muscle biopsies (See 
Figure). A biopsy of the Vastus Lateralis muscle will be performed using the Bergstrom 
technique.  Subjects will be placed in a supine position and the skin will be cleansed with 
chlorhexidine solution.  After a sterile drape is placed over the skin, local anesthesia will 
be administered using lidocaine.  The skin will be incised (approximately 0.5 cm) with a 
#11 scalpel, and the Bergstrom needle will be inserted into the Vastus 
Lateralis.  Approximately 150 mg of muscle tissue will be obtained under suction. After 
the biopsy, pressure will be applied to stop bleeding and the skin will be closed with steri-
strips (suture(s) if allergy to steri-strips).  A sterile dressing will be applied.  An MD, DO, 
NP, or PA will conduct this procedure. This procedure is well-tolerated in older adults. 
Subjects will not perform physical exercise 48 hours prior to the muscle biopsy to help 
prevent acute effects of exercise on muscle lipid and the other primary outcomes. Muscle 
specimens will be trimmed of any visible adipose and connective tissue using a dissecting 
microscope and processed. The following assays will be performed on the muscle biopsy 
specimens: 
 
1. Mitochondrial respiration will be measured in permeabilized muscle fibers 
from vastus lateralis biopsies using the Oxygraph 2K system (Oroboros, 
Innsbruck, Austria). This approach permits a comprehensive evaluation of 
mitochondrial respiratory function upon titration with ETC complex-specific 

 
 
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inhibitors and substrates (56). We assess complex I+II supported respiration and 
palmitate ß-oxidation supported respiration in separate assay protocols. 
Submaximal and maximal, coupled and uncoupled respiration will be evaluated 
along with coupling control ratios. This technique is well established in our 
laboratory (16, 19, 35) and we have conducted respirometry assays in over 500 
muscle biopsy specimens in recent years.  
 
2. Mitochondrial H2O2 emission will be measured as an index of redox state in 
permeabilized muscle fiber bundles by real time monitoring of Amplex Red 
oxidation using a spectrofluorometer, equipped with a thermojacketed cuvette 
chamber (Horiba Jobin Yvon, Edison, NJ). Dr. Coen has implemented the 
permeabilized muscle fiber method to quantify H2O2 emission rates as an index 
of cellular redox state (5) in over 400 biopsies to date with a high degree of 
within-sample reproducibility (CV<10%). 
 
3. Calcium retention capacity of mitochondria will be determined by 
spectrofluorometry. Change in free Ca2+ in the cuvette during mCa2+ uptake 
will be calculated using the known Kd for Calcium Green 5N and the equations 
established by Tsien (117) for calculating free ion concentrations using ion-
sensitive fluorophores. Dr. Coen has implemented the permeabilized muscle 
fiber method to quantify H2O2 emission rates as an index of cellular redox state 
and calcium retention capacity (5, 6) in over 400 biopsies to date with a high 
degree of within-sample reproducibility (CV<10%). Assays will be conducted 
by trained technicians in a blinded manner. 
 
4. Muscle protein synthesis and breakdown rate will be determined. Total and 
individual protein synthesis will be assessed in muscle biopsy specimens using 
deuterium oxide (D2O) according to their previously published procedures in 
humans (4, 103). 50mg of muscle biopsy specimen will be fractionated into 
myofibrillar, cytosolic and mitochondrial protein fractions according to their 
previously published procedures (30-32, 76). After protein hydrolysis 
derivatized amino acids will be analyzed on an Agilent 7890A GC coupled to 
an Agilent 5975C MS (30-32, 76). To assist in the determination of protein 
turnover during non-steady state conditions, the group recently established a 
mathematical model to determine rates of synthesis and breakdown using 
changes in enrichment over time (77).  
 
5. Multidimensional 3D shotgun lipidomics will be conducted. Briefly, lipids will 
be extracted from N2-frozen biopsy sample (20mg) by a modified Bligh and 
Dyer method (54). Internal standards will be used to quantify individual 
molecular species of lipid classes (54).. Mass spectrometry-based lipidomics 
will be performed on a TSQ Vantage QpQ mass spectrometer equipped with an 
Triversa Nanomate automated nanospray device and operated with Xcalibur 
software (69). Identification and quantification of individual molecular species 

 
 
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will be performed using an automated software program. This powerful 
approach will enable us to analyze over 30 lipid classes and hundreds to 
thousands of individual lipid species collectively representing more than 95% 
of the total content of the lipidome in human muscle. Total content and 
individual species of sphingolipid will be quantified. Cardiolipin will be 
quantified as an index of mitochondrial content as will oxidatively modified 
phospholipids malondialdeyde and 4-hydroxy-2,3-trans-nonenal.  
 
6. Immunoblot for activation of protein synthesis/degradation pathways. Key 
mediators of protein synthesis (AKT/P-AKT, P-mTORC2, P-4EBP, P-eIF4B 
P-S6K, P-S6), autophagy/proteasome mediated breakdown (P-FOXO3a, 
MuRF-1, Atrogin-1, LC3B1/2, Beclin-1, p62), and mitochondria content 
(OXPHOS) among others will be quantified by immunoblot with validated 
antibodies and +/- controls. This is a standard assay that is routinely performed 
in our group. 
 
7. Histochemical analysis for fiber type and muscle lipid will be determined by 
Oil red O staining intensity and cross-sectional area will be determined in type 
I and type II myocytes performed on serial sections using a modified version of 
methods previously used in our laboratory (14, 17). Other histological staining 
may also be performed, including but not limited to SDH and Glycogen. 
 
2Adipose Tissue Biopsy: This is an optional procedure that the participant will consent to 
if they would like to have it done. After vital signs are obtained, subjects will be placed in 
a supine position. The abdominal skin 6-10 cm lateral to the umbilicus will be cleansed 
with chlorhexidine. A sterile drape is placed, and the skin and adipose tissue will be 
anesthetized using Tumescent lidocaine. Depending on adipose deposits available and the 
clinician’s judgment, either a 3-4 mm Mercedes Liposuction needle, 13g Yale needle, or a 
4-6 mm Bergstrom needle will be inserted for aspiration of approximately 1000 mg of 
adipose tissue. Once the sample is aspirated, the needle will be removed. In the instance 
where the use of a needle under aspiration yields inadequate amounts of adipose tissue, 
additional adipose tissue may be collected using forceps and a scalpel to excise visible 
superficially located subcutaneous adipose tissue. The incision will be closed with steri-
strips (suture(s) if allergy to steri-strips), and sterile dressing will be applied. An MD, DO, 
NP, or PA will conduct this procedure.   
 
3Imaging Assessments (DEXA, IMAT, ATPmax and IMCL) previously defined in 
PreBed Rest Phase.   
 
4We will mitigate risk of VTE with daily passive exercise and through prophylactic 
anticoagulation with low-molecular weight heparin.  Daily passive exercise will be given 
throughout bed rest.  Low-molecular weight heparin will be given throughout bed rest, 
except on days when patient will undergo biopsy. 
 

 
 
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5Participants will be instructed to use an incentive spirometer 5-10 times per day (every 1-
2 hours) during bedrest to help clear their lungs.  
 
6Hyperinsulinemic-euglycemic clamp: The glucose clamp is the gold standard for 
measurement of insulin sensitivity and will be performed as previously described by our 
group (3, 15, 27, 29). After an overnight 8-hour fast, an intravenous catheter will be placed 
in the vein for infusion of insulin and glucose.  A second catheter will be placed in the vein 
of the contra-lateral arm for blood withdrawal. After baseline blood is collected, a primed 
(210 mg/m2), continuous (2 mg/min/m2) infusion of U13C glucose will be initiated for 
assessment of endogenous glucose production. A 2-step euglycemic clamp will be started 
with a 2-hour infusion of insulin (Humulin-R) at 10-30 mU/m2/min, followed by 2 hours 
at 80-100 mU /m2/min.  Plasma glucose will be measured at ~5 min intervals via NOVA 
StatStrip Glucose Meter. Plasma glucose will be allowed to either decrease to ~90 mg/dl 
or will be increased to ~90 mg/dl in each participant depending on the fasting blood glucose 
levels. Euglycemia (~90 mg/dL) will be maintained with a variable 20% dextrose infusion 
enriched with U13C glucose. Rates of glucose disposal (M) and endogenous glucose 
production (EGP) will be calculated by non–steady-state equations based on plasma U13C 
glucose enrichment determined by gas chromatography mass spectrometry. A baseline 
blood sample will be collected prior to -30 mins. Then three blood samples will be collected 
for determination of plasma insulin, free fatty acid (FFAs), C-Peptide, and glucose 
concentrations prior to the insulin infusion, and at the beginning of each steady state and 
every 10 minutes during the two steady state portions of the clamp which will last 
approximately 4 hours from the time at which the insulin infusion is initiated (-30min, -
20min, -10min, 0min, +30min, +60min, +100min, +110min, +120min, +220min, 
+230min, and +240min) . Participants will complete a 24-hour urine collection on days 
where hyperinsulinemic-euglycemic clamps are run. Total blood volume during this 
procedure will be approximately 192 mls. Bedrest ends after the completion of the 
hyperinsulinemic-euglycemic clamp on Day 10. 
Calculations: Hepatic glucose production is expected to be completely suppressed at this 
level of insulin, even in diabetic subjects.  In this case, peripheral glucose uptake (Rd) 
should equal the glucose infusion rate (GINF) during steady state (the last 30 minutes of 
each step of the clamp) after correction for urinary glucose loss.    
That is: - Rd = Steady State GINF - urinary glucose loss 
Insulin sensitivity (SI) will be calculated using the formula: SI = Rd / (steady state insulin 
level - basal insulin level) where steady state insulin equals the average insulin 
concentration during the last 40 minutes of the clamp and basal insulin equals the average 
insulin level in the 30 minutes before starting the insulin infusion.   
Fasting and insulin-suppressed FFA [IS-FFA] will be measured with an ultrasensitive FFA 
assay from Wako. Glucose disposal rate [GDR] will be expressed as mg/kg FFM 
[DEXA]/minute. 
Metabolic flexibility of substrate oxidation 

 
 
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Before and during the steady state of the clamp, we will measure substrate oxidation using 
a MAX I  Metabolic Cart (AEI Technologies, Pittsburgh, PA) to measure ‘metabolic 
flexibility’ as described by Kelley and Mandarino (62). 
 
7A blood collection will be done to test the participant’s hemoglobin and hematocrit levels 
on Day 7 and Day 10. If the hemoglobin level is < 10.0 g/dL for females or < 11.0 g/dL for 
males on either day, the medical provider may repeat the tests and/or withdraw the 
participant from the study. 
 
8Administration of D2O. 25ml of 99% D2O will be consumed 2x/day for the first week 
following bed rest and 25 ml once a day for the next 3 weeks, with the last dose being given 
the day before week 4, visit 16.This regimen will provide a stable, continuous precursor 
pool enrichment of body water. 
 
9A number of health questionnaires will be administered following bed rest, including: Life 
Space, Lifestyle, Functional Status, Pittsburgh Fatigability Scale. 
 
10Resting Metabolic Rate/Respiratory Quotient (RMR/RQ) will be determined that 
morning in the fasting state. 
 
11An orthostatic hypotension assessment will be completed once participants become 
ambulatory. This will be completed by measuring blood pressure while in lying down, 
sitting, and standing positions. The assessment will be completed to ensure participant 
safety prior to being discharged from TRI. 
 
Phase IV-Ambulatory/Exercise Recovery Program 
 
Exercise Recovery. The exercise recovery program will be supervised by a certified 
exercise physiologist and will take place at the TRI exercise training facility. Exercise 
compliance will be monitored carefully. We are examining the muscle specific effects of 
the recovery exercise program on the muscle groups of interest. We will use an exercise 
recovery program that has demonstrated substantial increases in quadriceps muscle size 
and strength in elderly people (37, 40, 105). During the 4 weeks of recovery, participants 
will perform a combination of aerobic and resistance exercise training with a certified 
exercise physiologist 3 days per week at the TRI exercise training facility. Exercise 
compliance and adherence will be monitored carefully. Aerobic training will be 30 minutes 
of brisk walking, jogging, cycle ergometry and will be at 60-80% of post bed rest VO2max 
peak HR.  Participants will wear a Heart Rate Monitor during Aerobic exercise sessions. 
Walking will be the primary mode of aerobic exercise given its widespread popularity and 
ease of administration across a broad segment of the older adult population. Three sets of 
8–12 repetitions of each resistance exercise will be performed to target the major lower 
body muscle groups, including the vastus lateralis of the quadriceps. 
 

 
 
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Ambulatory Recovery. Participants in the ambulatory recovery group will not receive any 
exercise intervention or advice on exercise. Rather these participants will return to their 
regular daily routine that they engaged in prior to the bed rest intervention. 
 
Participants in both Exercise and Ambulatory Recovery will be admitted to TRI the 
evening before Week 4 / Visit 16 for an overnight stay. The participants will be served 
dinner and type 2 diabetics will be asked to bring their own home glucometer to test 
glucose.  
 
The following table summarizes the schedule of assessment during Ambulatory/Exercise 
Recovery Phase. Descriptions of assessments can be found above (Phase III). 
 
 
 
 
 
 
Week 
Week 1 
Week 2 
Week 3 
Week 4 
Visit 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
Day of Week 
F 
M 
T 
Th 
F 
M 
T 
Th 
F 
M 
T 
Th 
F 
M 
T 
F 
Window Days 
+1 
+1 
+1 
+1 
+1 
+1 
+1 
+1 
+1 
+1 
+1 
+1 
+1 
+1 
+1 
+3 
Fasting Muscle 
Biopsy/Blood Draw1 
  
  
  
X*^ 
  
  
  
X*^ 
  
  
  
X*^ 
  
  
  
X*^ 
Fasting Adipose 
Biopsy2 (optional) 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
X*^ 
24-Hour Urine 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X*^ 
Glucose Clamp - 
4Hr6 with RMR 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
X*^ 
Vital Signs 
X* 
X* 
X* 
X*^ 
X* 
X* 
X* 
X*^ 
X* 
X* 
X* 
X*^ 
X* 
X* 
X* 
X*^ 
Medication/Supplem
ent Intake & Changes 
X* 
X* 
X* 
X*^ 
X* 
X* 
X* 
X*^ 
X* 
X* 
X* 
X*^ 
X* 
X* 
X* 
X*^ 
Adverse Events 
X* 
X* 
X* 
X*^ 
X* 
X* 
X* 
X*^ 
X* 
X* 
X* 
X*^ 
X* 
X* 
X* 
X*^ 
Metabolic Body 
Weight16 
  
  
  
X*^ 
  
  
  
X*^ 
  
  
  
X*^ 
  
 X*^ 
  
X*^ 
BIODEX/Strength 
Testing 
  
  
  
  
  
  
  
  
  
  
  
X*^ 
  
  
  
  
Handgrip Strength 
  
  
  
  
  
  
  
  
  
  
  
X*^ 
  
  
  
  
SPPB 
  
  
  
  
  
  
  
  
  
  
  
X*^ 
  
  
  
  
Step Test 
  
  
  
  
  
  
  
  
  
  
  
X*^ 
  
  
  
  
Questionnaires2 
  
  
  
  
  
  
  
  
  
  
  
  
  
X*^ 
  
 
DEXA 
  
  
  
X*^ 
  
  
  
X*^ 
  
  
  
X*^ 
  
  
  
X*^ 
MR Assessments 
(ATPmax/IMCL/IM
AT) 
  
  
  
X*^ 
  
  
  
X*^ 
  
  
  
X*^ 
  
  
  
X*^ 

 
 
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Accelerometer 
Handout 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
X* 
X^ 
Accelerometer 
Collection 
  
  
  
  
  
X* 
  
X^ 
  
  
  
  
  
  
  
  
VO2max Test 
  
  
  
  
  
  
  
  
  
  
  
  
  
X*^ 
  
 
Cardio Exercise 
Program 
X* 
X* 
X* 
  
X* 
X* 
X* 
  
X* 
X* 
X* 
  
X* 
X* 
X* 
  
Strength Exercise 
Program 
X* 
X* 
X* 
  
X* 
X* 
X* 
  
X* 
X* 
X* 
  
X* 
X* 
X* 
  
Deuterated H2O 
adminstration8 
X*^ 
X*^ 
X*^ 
X*^ 
X*^ 
X*^ 
X*^ 
X*^ 
X*^ 
X*^ 
X*^ 
X*^ 
X*^ 
X*^ 
X*^ 
 
400m Walk Test 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X*^ 
 
 
* = only on this visit for exercise cohort 
^ = only on this visit for ambulatory cohort 
 
Phase V – Optional Resistance Training. 
Following Phase IV of the study, to expedite complete recovery, subjects may attend three 
resistance exercise training sessions per week over a four-week period, for a total of 12 
visits. Resistance exercise training will include weight training of all major muscle groups. 
Subjects will perform three sets of eight repetitions at 70-80% of their 1 RM for each 
exercise, which will include upper body and lower body muscle groups. During all exercise 
sessions, subjects must be supervised by qualified personnel with the appropriate training 
to identify if the subject is under any type of distress. Study assessments will also be 
performed during the 4-week resistance training according to the table below: 
 
Assessment 
Resistance Training (Optional) 
Week 1 
Week 2 
Week 3 
Week 4 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
Vital Signs 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medication/supplement 
intake and changes 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Adverse events 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Metabolic Body Weight16 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Resistance Training1 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
BIODEX/Strength Testing 
X 
 
 
 
 
 
 
 
 
 
 
X 
Handgrip Strength 
 
 
 
 
 
X 
 
 
 
 
 
X 
SPPB 
 
 
 
 
 
X 
 
 
 
 
 
X 
Step Test 
 
 
 
 
 
X 
 
 
 
 
 
X 
Questionnaires2 
 
 
 
 
 
X 
 
 
 
 
 
X 
Accelerometer Collection 
 
X 
 
 
 
 
 
 
 
 
 
 
 

 
 
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Before and after any exercise activities are performed, the participant’s blood glucose will 
be checked (type 2 diabetics only) using their home glucose monitor or glucose monitor at 
TRI. Blood glucose will be measured prior to and immediately following each exercise 
session for safety.  If blood glucose is > 300mg/dl, study team will notify TRI provider 
immediately for review of glucose values, as well as medications.  If the glucose values 
are consistently > 400mg/dl, participant will be instructed to follow up with their primary 
care physician. If blood glucose is < 100mg/dl, a snack will be given, and the participant 
will be allowed to exercise. Self-monitored blood glucose measurements should be 
checked anytime a patient has symptoms of hypoglycemia or hyperglycemia.   
 
1Resistance training will include weight training of all major muscle groups 3 times per 
week for 4 weeks. 
 
2A number of health questionnaires will be administered during the ambulatory recovery 
phase, including: Life Space, Lifestyle, Functional Status, Pittsburgh Fatigability Scale. 
CHAMPS will be administered. 
 
Study Duration 
 
Estimated start date for enrollment start is August 1st, 2019. Enrollment is projected to take 
~3 years. Patients will be studied over a ~16-week period. The study is funded by the 
National Institute on Aging until 05/31/2023. 
 
Materials of Human Origin:  Collection, Preparation, Handling and Shipping 
 
Materials of human origin will be collected in the manner described in the specific study 
visits section of this protocol.   
 
Biospecimen samples will be stored in ultralow temperature freezers and liquid nitrogen 
dewars or other storage units located at the TRI Laboratory Room 2404. The TRI facility 
is secured via key card and equipped with a back-up generator system. Laboratory 
personnel in the facility have 24/7 key-controlled access to the laboratory.  Chain of 
custody of biospecimen samples is maintained through requisition forms and in the 
StarLIMS database. Specimen tubes are coded, and specimen requests and distribution are 
documented. 
 
The plasma/serum samples will be stored indefinitely, or until a sample is fully used. All 
biological specimens will be stored without identifiers or linkage codes.  
 
After study aims have been achieved and study related endpoints have been measured and 
analyzed, any remaining biospecimens will be stored at the TRI Laboratory Room 2404 
and will then be considered as “archived biospecimens.”  Archived biospecimens will be 
used for any additional hypothesis-related experimentation or testing for the purposes of 

 
 
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this study, consistent with the original aims, which cannot be predicted at the time the 
protocol is developed due to the evolving nature of scientific exploration.   
 
Additionally, archived biospecimen samples may be stored indefinitely for future research.   
Archived biospecimens could be used for separate research by both AdventHealth 
Orlando scientists and scientists outside of AdventHealth Orlando. This would be allowed 
for research of any type (without limitation to disease, process, or research methods) if it 
has scientific merit as determined by the Principal Investigator, with an additional review 
by the respective Program Director.  For research outside of AdventHealth Orlando, a 
Material Transfer Agreement will be obtained, which will govern the transfer and chain of 
custody of the biospecimens outside of AdventHealth Orlando. 
 
Study Outcome Measures (Endpoints) 
The primary outcomes, including leg lean mass, protein synthesis rate, and mitochondrial 
respiration, will be measured at 5 time points (immediately following bed rest, and on 
weeks 1, 2, 3, and 4 of Recovery Phase). 
 
Data Management and Quality Plan 
 
Data De-identification 
Participants will be enrolled using Cerner’s Patient Protocol Manager; the application 
assigns each participant a unique participant identifier, or “PID”.  This PID is a code 
consisting of a combination of numerals and letters, which serves as the identifier for this 
participant for this research study and links them back to their hospital medical record and 
their protected health information (PHI).  Access to the “link” between the PIDs, the PHI, 
and to the clinical data are only granted to the clinical research team as assigned on the 
Delegation of Authority Log.   All the clinical research data is recorded in a de-identified 
fashion onto source documents, which is then transcribed into the electronic case report 
forms, (CRF). The CRF is used for storage (a database) and facilitates analysis.  Clinical 
data generated by research devices also uses the PID, and once the data has been 
transformed into interpretable results it is stored into the clinical research database. Both 
storage locations are secured and only assessable to the assigned clinical research team.  
The “link” will not be used to re-identify participants except in the event of a serious 
adverse event (SAE) requiring “unblinding” to treat the participant.  The “link” will be 
stored in the Patient Protocol Manager, where only the TRI research team has access. These 
secure databases are stored/accessed on the AdventHealth Orlando password-protected 
computer network. No one outside of AdventHealth Orlando investigators or researchers 
will have access to the databases.  
    
Data Confidentiality, Storage, and Retention 
The identity and personal health information will be kept confidential to the extent 
permitted by the applicable laws and/or regulations and will not be made publicly available. 
If results of this study are published or presented, the identities will not be revealed. 
Confidentiality will be maintained during and after the study.  This information is also 

 
 
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included in the informed consent, which is discussed with the participant prior to 
enrollment. 
 
Study documentation and paperwork will be stored in our locked medical records 
room.  Some records will be stored as electronic records.  This data will be safeguarded so 
that only those on the research team have access to the clinical data. The electronic data is 
maintained by AdventistHealth Information Technology (AIT) security controls.   
 
The duration of study data retention will be determined by governing FDA regulations 
and/or sponsor contract mandate (if applicable). TRI retention policy is maintained in the 
Records Management Policy.  Electronic de-identified data will be kept indefinitely in our 
database. 
 
Data Quality  
Data quality control will occur according to our SOPs on Data Entry, Quality Control 
Procedures and Query Management.  All data will be entered into an electronic data capture 
(EDC) system and checked against the paper source for accuracy by a second party (Data 
Entry SOP) and errors resolved through the Query Management SOP.  Ten percent of the 
data points will be routinely checked at the beginning, middle, and close of a study for 
quality control (Quality Control SOP).  Finally, all critical endpoints (as determined by the 
PI or Sub-I) will be assessed using quality control analyses.  The data will be exported into 
the study database.   
 
The device data will directly be imported into spreadsheets or entered and confirmed into 
the EDC.  Data from the VO2max, activity monitors, and DEXA data are exported directly 
into spreadsheets for further calculations. Data for each of the endpoint analyses will be 
imported into an SAS database linked with a participant ID.  
 
Data Sharing (outside of AdventHealth Orlando)  
This is a collaboration partnership between TRI, the Sponsor, and its affiliates. Certain data 
elements will need to be shared along with the biospecimen samples (primarily satellite 
cell cultures). Should archived biospecimens be needed for research outside of 
AdventHealth Orlando and certain data elements that are connected to the archived 
biospecimen samples be needed to conduct the research, then Data Use Agreement(s) will 
be obtained.  The Data Use Agreement(s) will identify the purpose for data sharing, the 
specific data elements to be shared, and will govern the sharing of data related to this study.  
Data will be de-identified, but a link/code is managed within an electronic research 
management system and maintained by a study coordinator.  
 
Sample Size Determination 
 
Fifteen (15) participants are expected to complete the study after conservatively estimating a 25% 
drop-out rate. This will be sufficient to provide 80% power to detect a significant mean difference 
in leg lean mass between groups during recovery, based on previously published data (REF), and 

 
 
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from out bed rest and recovery study in older adults (recovery in leg lean mass over 4 weeks (n=20; 
mean ± SD) = +1.2 ± 0.5 kg). Sex will be explored as a biological variable, however, based on 
data from our previous bed rest and recovery study we do not expect a difference in rate of recovery 
between men and women (n=10 men +1.1 kg, n=10 women +1.0 kg lean mass recovery). The 
sample size will also provide 80% power to detect a significant mean difference of 0.38 AU in 
muscle mitochondrial OXPHOS protein expression between groups during recovery. The power 
calculations are based on two-sample t-tests for mean difference with unequal variance and a P-
value of 0.05. 
 
Statistical Analysis Plan 
 
The primary outcomes, including leg lean mass, protein synthesis rate, and mitochondrial 
respiration, will be measured at 5 time points following 10 days of bed rest. 2x5 repeated-measures 
ANOVA will be performed on the dependent variables as a function of intervention (2 levels: EX 
and CON groups) and time (3 levels: 5x biopsy timepoints). We will also fit mixed models (fixed 
and random effects) to interrogate the longitudinal data. This will allow us to better account for 
the longitudinal nature of the data and to explore the influence of other potential predictors of 
changes in muscle mass. In particular, the relationship between change in IR, protein synthesis and 
mitochondrial function will be explored. Since many of these outcome variables are from biopsies, 
e.g., various mitochondria parameters will be correlated with one another, covariance will be 
explored, and we will also use the relative change measures of these as a bivariate outcome. Sex, 
race and ethnicity as biological variables will also be explored. Sex will be explored as a biological 
variable, however, based on data from our previous bed rest and recovery study we do not expect 
a difference in rate of recovery between men and women (n=10 men +1.1kg, n=10 women +1.0 
kg leg lean mass recovery). 
 
Potential Risks and Benefits 
 
Potential Benefits 
 
Participants will likely receive no direct benefit from taking part in this research study. 
 
Potential Risks 
 
 
Blood draws and/or IV (lab samples, e.g.). There is a risk of pain, vasovagal syncope, 
hematomas, and/or infection at the blood draw site or IV insertion site (low risk of serious 
AEs).   
 
DEXA scan. There is a very small risk of cancer with excessive exposure to any radiation.  
There are also risks for unborn children associated with radiation exposure. The radiation 
dose from one scan is less than a chest x-ray.  There are six scans in this study; the total 
radiation is less than 4 mrem, or less than the same average amount a person would receive 
over 3 days in America. 
 

 
 
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Muscle Biopsy. There is a risk of pain from the local anesthesia, and a risk of bruising, 
bleeding (hematoma) and infection at the site of the biopsy. In addition, there is a risk of 
skin irritation due to the steri-strips and dressing. Local sensory loss may occur by cutting 
a subcutaneous sensory nerve (<1 in 100), which is almost always temporary but 
occasionally can become permanent. On rare occasion (<1 in 1000) bleeding, requiring 
hospitalization, may occur. The procedures are well tolerated when performed properly. 
 
Optional Adipose Tissue Biopsy. There is a risk of pain from the local anesthesia, and a 
risk of bruising, bleeding and infection at the site of the biopsy. In addition, there is a risk 
of skin irritation due to the steri-strips and dressing.  
 
Oral Glucose Tolerance Test. There is a risk of pain, hematomas, and/or infection due to 
venipuncture (low risk of serious AEs).  Nausea from glucose ingestion may occur. 
 
 
Biodex/Strength testing/Exercise/Maximal Oxygen Consumption (VO2max). There is a 
risk of changes to blood pressure, irregular, fast or slow heart rhythm, fainting, and in rare 
instances (<0.01%) heart attack, stroke, or death with exercise testing, at similar rates to 
exercising during daily life.   
 
Activity/Heart Rate Monitoring. There are no risks associated with the wearing of 
monitors, however the armband/chest band that holds the monitor in place may be irritating 
to the skin for some subjects.  Participants with nickel allergies may have irritation at the 
site of the monitor. 
 
Bed Rest Intervention. The 10-day bed rest intervention will result in a mild 
deconditioning that may increase fatigability and lower muscle strength (10-20% decrease) 
and mass (-3% approximately) and increased body fat mass of participants. Individuals 
with prolonged immobility are at risk for venous thromboembolism (VTE). There is also a 
risk of pressure ulcers – extrinsic pressure greater than capillary perfusion pressure 
(30mm/Hg) for a prolonged period of time results in ischemia to the affected tissue. Both 
hyperglycemia and hypoglycemia are risks for hospitalized patients with T2D and are 
associated with adverse health outcomes. However, these risks are mitigated by close 
clinical monitoring during the bed rest intervention (VTE prevention and glycemic control 
plans) and completion of the exercise recovery program. The participants will be informed 
that they may abort the study at any time. There are no alternative procedures/methods 
available with less risk or cost that provide comparable information to that obtained by the 
procedures described in this proposal. 
 
Enoxaparin. There is a risk of bleeding, nausea, diarrhea, fever, swelling of the hands or 
feet, and injection site reactions including swelling, pain, bruising, and redness. 
Concomitant medications with known contraindication or interaction with low-molecular 
weight heparin (including anti-platelet agents, non-steroidal anti-inflammatory drugs) will 
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RMR/RQ.  There is no physical risk associated with RMR/RQ. Other risks include a 
feeling of claustrophobia experienced by some subjects while under the transparent 
“hood”. 
 
Magnetic Resonance Imaging and Spectroscopy. There are no known biological risks 
associated with magnetic resonance imaging and spectroscopy. Some short-term 
discomfort may be experienced.  The short-term risks associated with MRI are minimal, 
but include heating, loud noises and claustrophobia.  There are some people who should 
not undergo MRI; the contraindication is largely based on the presence of metal objects 
within a person (i.e. pacemaker, aneurysm clip, metal fragments, etc.).   
 
Hyperinsulinemic-euglycemic clamp. There is a risk of hypoglycemia (low blood sugar) 
during the clamp.   
 
Mitigation of Risks 
 
Blood draws and/or IV (lab samples, e.g.). All venipuncture will be conducted by 
qualified staff using aseptic techniques. Total blood volume throughout the entire study 
will be approximately 550 mls.  
 
Muscle Biopsy. Muscle biopsies will be conducted by qualified staff following 
institutional policies and procedures including sterile techniques and sterile dressing to the 
site. Prior to the biopsies, participants will be asked for allergies including to lidocaine and 
the use of anticoagulants. Subjects allergic to lidocaine or that used anticoagulants in 
clinically significant amounts will be excluded. Sutures will be used to close the site, for 
those with a known allergy to steri-strips. 
 
Optional Adipose Tissue Biopsy. Adipose biopsies will be conducted by qualified staff 
following institutional policies and procedures including sterile techniques and sterile 
dressing to the site. Prior to the biopsies, participants will be asked for allergies including 
to lidocaine and the use of anticoagulants. Subjects that have an allergy to lidocaine or have 
used anticoagulants in clinically significant amounts will be excluded. Sutures will be used 
to close the site, for those with a known allergy to steri-strips. 
 
Oral Glucose Tolerance Test. All venipuncture will be conducted by qualified staff using 
aseptic technique.   
             
Biodex/Strength testing/Exercise/Maximal Oxygen Consumption (VO2max). Prior to 
conducting any exercise testing, a history and physical including ECG will be conducted 
and will be used to determine whether a subject is clear to participate in exercise testing. 
An MD/DO/NP/PA will be present during exercise testing for subjects who are at risk 
according to the American College of Sports Medicine (ACSM) Guidelines. ECG, blood 
pressure and heart rate may be monitored during the test. 

 
 
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Activity/Heart Rate Monitoring. Participants with nickel allergies will not be required to 
wear the monitor.  
 
Bed Rest Intervention. During bed rest, the participants will be closely monitored by CRU 
nursing staff and medical oversight will be provided by Dr. Richard Pratley, a 
physician/scientist with over 30 years of experience in conducting clinical investigations 
in diabetes and obesity. 
 
Venous Thromboembolism. (VTE) risk mitigation. Individuals with prolonged 
immobility are at risk for VTE. We have used the American College of Chest Physicians 
(ACCP) Evidence Based Clinical Practice Guidelines in understanding the risk profile of 
enrolled participants for the development of venous thromboembolism as well as the risk 
of major bleeding with thromboprophylaxis. 
  
According to the ACCP, the Padua Risk Prediction score provides the best available model 
for assessing risk – based on the Padua Risk Prediction model, [which assigns 3 points to 
reduced mobility, 1 point to Age ≥ 70, and 1 point to Obesity (BMI ≥ 30)] the majority of 
participants in this study will have a score ≥4, which qualifies as “high risk of VTE”. We 
will mitigate risk of VTE in our participants by employing a comprehensive prophylactic 
plan that includes anticoagulation with low-molecular weight heparin and daily passive 
exercise throughout bed rest. 
  
Non-treated baseline risk of bleeding was defined as 0.4% based on the control arm of trials 
of thromboprophylaxis in medical patients. Subsequent analysis of any anticoagulant vs. 
none to prevent VTE and low-molecular weight heparin (LMWH) vs. low-dose 
unfractionated heparin (LDUH) demonstrate a significant reduction in fatal pulmonary 
embolisms (PEs) and a reduction in symptomatic deep venous thrombosis (DVT). The 
effect of anticoagulation on major bleeding was not statistically significant (and relatively 
low, from baseline risk of 4 per 1,000 to 1 more per 1,000). There was no statistically 
significant effect of anticoagulation on all-cause mortality. 
  
In addition to the risk of bleeding described above, heparin-based anticoagulation is 
associated with a rare entity called heparin induced thrombocytopenia (HIT). As per the 
ACCP Evidence Based Guidelines, medical patients who are only receiving LMWH will 
not have routine platelet count monitoring and the associated risk of HIT is <0.1%. 
 
Mechanical methods of thromboprophylaxis were evaluated and associated with an 
unfavorable rate of skin issues and less risk reduction for symptomatic DVT as compared 
to anticoagulation in high risk patients. Intermittent pneumatic compression devices had 
not been studied in hospitalized medical patients; data reviewed through a meta-analysis 
failed to demonstrate a beneficial effect on mortality or PE but reduced the risk of DVT 
primarily in surgical patients. Thus, compression devices will not be utilized. 
 

 
 
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Therefore, we will mitigate risk of VTE in our high-risk participant population through 
prophylactic anticoagulation with low-molecular weight heparin given its safety profile 
and advantageous dosing frequency (qd vs. BID/TID for LDUH). Participants will receive 
enoxaparin 40mg subcutaneous injection, daily while immobile, with dose held only on the 
day of biopsies to minimize risk of bleeding or oozing intra- and post-procedurally. We 
will employ the daily passive exercise, throughout bed rest. 
 
Every effort to prevent a study-related injury will be taken.  If a DVT is suspected, the 
participant will be assessed by a provider, withdrawn from the study, and instructed to seek 
emergency treatment immediately. Sources: Prevention of VTE in Nonsurgical Patients - 
https://journal.chestnet.org/article/S0012-3692(12)60124-X/fulltext 
 
HIT - https://journal.chestnet.org/article/S0012-3692(15)31496-3/pdf 
 
Pneumonia Prevention 
Participants will also use an incentive spirometer 5-10 times (every 1-2 hours) daily to 
prevent pneumonia. 
 
Glycemic management protocol. Hyperglycemia and hypoglycemia are risks for 
hospitalized patients with T2D and are associated with adverse health outcomes. To 
mitigate this risk patients with HA1C < 8.0% will be recruited. In addition, a glycemic 
management protocol will be administered for type 2 diabetics by the study physician. 
Capillary blood glucose will be measured four times a day, before each meal and at 
bedtime. If two consecutive results of > 240mg/dl are recorded, the study physician will 
review and change the patient’s current treatment regimen (DPP-4 inhibitors, sulfonylureas 
and/or Metformin) to prevent further hyperglycemia. If two consecutive results of 
>270mg/dl are recorded, the study physician may initiate insulin therapy. Insulin therapy 
will continue until the participant reaches a target glucose range of 140–180 mg/dL (7.8–
10.0 mmol/L). The participant’s treatment regimen and diet will be reviewed and changed 
as necessary to prevent hypoglycemia (<70 mg/dL), although this is not expected with 
medications like, DPP-4 inhibitors and/or Metformin. 
 
In addition, the following procedures, evaluations and/or assessments will be obtained 
daily during bed rest: 1) Medication/supplementation changes, 2) Vital signs consisting of 
body temperature, pulse rate (or heart rate), blood pressure, and respiratory rate will be 
monitored routinely. 3) Subjects will be interviewed to determine if any AEs/SAEs 
occurred in the last 24 hours. 4) Subjects will be offered medication to help mitigate reflux 
problems, constipation, and other issues associated with being supine. Following 10 days 
of bed rest, the post bed rest recovery will consist of assisting the participant with 
ambulation and ensuring the participant is capable of showering, walking and dressing 
without assistance. The participant may stay an extra night at the study site for further 
recovery if it is deemed medically necessary by the study physician. A follow up phone 
call will be made to ensure the subject is ambulatory, remind the subject about the exercise 

 
 
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schedule, and to inquire about any adverse events. Participants receive supervised exercise 
recovery program. 
 
Skin Care. Turning and adjustment in bed will be the primary preventative measure. Skin 
assessments will be performed twice per day during the bedrest phase of the study.  If there 
is a loss of skin integrity a skin management plan will be employed as directed by the 
medical providers. 
 
RMR/RQ.  A member of the study staff will remain with the subject at all times to ensure 
his/her comfort. 
 
Magnetic Resonance Imaging and Spectroscopy. There will be a strict safety screening 
protocol, to ensure any people with contraindications are excluded from volunteering. 
There will be no diagnostic analysis associated with any of the MR sequences used in this 
protocol.  However, some of the MR images we obtain as part of this protocol may show 
incidental medical findings.  In the case where a medical abnormality is apparent on an 
image, the image will first be reviewed by an investigator on this protocol.  If the 
abnormality is confirmed, then the participant will be instructed to seek medical attention 
from their health care provider.   
Hyperinsulinemic-euglycemic clamp. In-house supervision by a qualified physician or 
mid-level provider at all times during the procedure. Constant monitoring of blood glucose 
during the procedure (every 5 minutes). Immediate availability of corrective measures (IV 
50% dextrose and/or glucagon). 
 
Provisions to Protect the Privacy Interest of Subjects 
 
Participants will be assigned unique identifiers for study-related records.   All precautions 
will be taken to make sure that only authorized individuals will access participant research 
records.  The collection of sensitive information about participants will be limited to 
minimum necessary to achieve the aims of the research, so that no unneeded sensitive 
information will be collected. 
                                                                                                                                    
 
Early Withdrawal of Subjects 
Investigator Withdrawal of Subjects 
 
The participation in this study may be stopped at any time by the study PI without the 
participant’s consent because: 
-The study Medical investigator thinks it necessary for subject’s health or safety; 
-Participant has not followed study instructions; 
-The TRI has stopped the study; or 
-Administrative reasons require the participant’s withdrawal. 
 

 
 
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Subject Request for Withdrawal from Study 
 
Participation in this study is voluntary. Participants may decide not to participate in this 
study or may withdraw from this study at any time without penalty or loss of benefits.  
 
Data Collection and Follow-up for Withdrawn Subjects 
 
Participants who request withdrawal or who are withdrawn by the PI from the study will 
have their data maintained in the research database.  This data and biospecimens from the 
withdrawn participant may be included in subsequent analysis. Participant withdrawal of 
biospecimens is not an option. 
 
Adverse Event Reporting 
Adverse Events 
 
An adverse event (AE) is defined as both an expected side effect that is of a serious nature, 
or an unexpected side effect/event regardless of severity. Each participant is evaluated for 
adverse events at every study visit.  Any event that is reported to the study staff and which 
meets the criteria of an adverse event will be documented as such and graded as to its 
attribution (unrelated to protocol, or possibly, probably, or definitely related to protocol) 
and severity (mild, moderate, or severe).  
 
Recording of Adverse Events 
 
At each contact with the subject, the investigator will seek information on adverse events 
by specific questioning and, as appropriate, by examination.  Information on all adverse 
events will be recorded immediately in the source document, and also in the appropriate 
adverse event module of the case report form (CRF).  All clearly related signs, symptoms, 
and abnormal diagnostic procedures results will be recorded in the source document. 
 
Notification of Adverse Events 
 
Adverse events will be reported according to AdventHealth IRB guidelines. 
 
Safety Monitoring Plan   
 
Safety Monitoring 
 
Research and safety data will be reviewed by the PI. This review will take place at regular 
meetings with the clinical coordinator and medical investigator where the safety labs for 
each new subject will be reviewed. Other items discussed will include: progress or adverse 
events occurring in the following: subject confidentiality, subject recruitment, and consent 

 
 
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process. All will monitor response to tolerance of and effectiveness of the exercise 
program.  
 
Progress reports, including patient recruitment, retention/attrition, and AEs from the 
reviews will be compiled into an annual report and will include a list and summarization 
of adverse events.  In addition, the annual report will address (1) whether adverse event 
rates are consistent with pre-study assumptions; (2) reason for dropouts from the study; (3) 
whether all participants met entry criteria; (4) whether continuation of the study is justified 
on the basis that additional data are needed to accomplish the stated aims of the study; and 
(5) conditions whereby the study might be terminated prematurely.  The annual report will 
be forwarded to the Advent Health IRB and the TRI. 
 
Data and Safety Monitoring Board (DSMB) or Equivalent 
 
A Data and Safety Monitoring Board (DSMB) will be assembled by the NIH Program and study 
investigators. The DSMB will meet at a regular cadence that will be decided by the members of 
the DSMB. The DSMB will provide oversight and monitoring to ensure the safety of participants 
and the validity and integrity of the data and will be empowered to provide recommendations 
related to all aspects of protocol integrity as well as factors affecting participant safety. We propose 
that the DSMB consist of members external to the institution, without conflicts of interest and with 
the following qualifications: 
1. An expert in the science of exercise and effects of physical activity. 
2. A physician who can lead the effort in monitoring the overall event rate of various adverse 
events (AE) and serious adverse events (SAE), as well as balance between intervention 
groups. 
3. An external statistician who can monitor data collection, critique analysis plans, request 
additional data analysis, and provide interpretation of results to the DSMB members. 
 
For the first DSMB meeting, the board will review the DSMB plan, study protocol, quality control 
measures, and analysis plans. Subsequent DSMB teleconferences will focus on participant 
recruitment, intervention session attendance rates, retention rates, adverse event (AE) and serious 
adverse event (SAE) summaries and classifications, data quality control, and response to previous 
DSMB recommendations. Additional information will be provided to the DSMB with break down 
by randomized group assignment and these will be reviewed in the closed session with the study 
statisticians.  Separate systems will be put in place to handle the report of AEs and SAEs to the 
Chair of the DSMB and the AdventHealth IRB at the study site in a timely fashion after 
adjudication. 
 
Data and Safety Monitoring Plan 
 
As this project has relatively few subjects, the study progress and safety will be reviewed 
weekly by the PI, one of the study physicians, and/or research coordinator including 
progress or adverse events occurring in the following: subject confidentiality, subject 
recruitment, and consent process. All will monitor response to tolerance of and 

 
 
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effectiveness of the intervention program.  Overall, data integrity and participant safety 
will be monitored and maximized by the study investigator, clinician, and research 
coordinator involved who are experienced at conducting bed rest and exercise trials and 
specifically in the methods of collection of biospecimens to study the molecular changes 
in response to an inactivity and exercise challenge.  
 
Monitoring Plan 
The DSMB and Study Investigators will be responsible for the following: 
1. Review the research protocol and plans for data and safety monitoring, including 
all proposed revisions. 
2. Review methodology used to help maintain the confidentiality of the study data and 
the results of monitoring by reviewing procedures put in place by investigators to 
ensure confidentiality. 
3. Monitor study design, procedures and events that will maximize the safety of the 
study participants and minimize the risks. 
4. Evaluate the progress of the study, including periodic assessments of data quality 
and timeliness, participant recruitment, accrual and retention, participant risk 
versus benefit, performance of the study site(s), and other factors that may affect 
study outcome. 
5. Consider factors external to the study when relevant information becomes 
available, such as scientific developments that may have an impact on the safety of 
the participants or the ethics of the study. 
6. Review serious adverse event documentation and safety reports and make 
recommendations regarding protection of the safety of the study participants. 
7. Evaluate and report on perceived problems with study conduct, enrollment, and/or 
data collection, including: 
8. Progress reports, including patient recruitment, retention/attrition, and AEs from 
the weekly reviews. 
 
In addition, the DSMB reports will address (1) whether adverse event rates are consistent 
with pre-study assumptions; (2) reason for dropouts from the study; (3) whether all 
participants met entry criteria; (4) whether continuation of the study is justified on the basis 
that additional data are needed to accomplish the stated aims of the study; and (5) 
conditions whereby the study might be terminated prematurely. 
 
In addition to the above, among the most comprehensive and effective method of 
monitoring risks in studies with relatively few subjects is a weekly and biweekly individual 
case that will be reviewed by the PI, one of the study physicians, and/or research 
coordinator including progress or adverse events occurring in the following: subject 
confidentiality, subject recruitment, and consent process. All will monitor response to 
tolerance of and effectiveness of the exercise program. Files will be kept in a locked file 
cabinet in the laboratory of the PI to assure individual privacy and confidentiality of subject 
records. The research coordinators will monitor the following items: the timeframe of 
recruiting subjects, quality of data being entered, any external factors relevant for the safety 

 
 
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of participants throughout the entire study and is also instructed to make the PI aware of 
all events, expected or unexpected. 
 
Ethical Considerations 
 
Participation in this study is voluntary. Subjects may decide not to participate in this study or may 
withdraw from this study at any time without penalty or loss of benefits. No vulnerable populations 
will be studied in this protocol.  
Sharing of Results with Subjects 
 
Copies of the participant’s testing results may be made available to the participants. 
In addition, the Principal Investigator will provide an overview of the study's 
outcome to the participant if he or she requests the information. 
 
Funding Source 
 
This study will be supported by a research program grant (RPG) from the National Institutes of 
Health, National Institute on Aging (R01 AG060153, P.I., Paul M. Coen, Ph.D.). The IRBNet # 
for this grant is 992354. 
 
Subject Stipends or Payments 
We have budgeted an honorarium of $3,550 for participating. In the event the participant does not 
complete all study visits or procedures, payments will be prorated.  
 
Participants will be given a Mastercard® as a means to receive payments for this study. The 
terms and conditions for this card will be provided for the participant for review. Payment will 
be requested within 3 business days from the completion of the study visits. 
Payments will be requested after completion of Bed Rest and Resistance Week 4 for a total of 2 
study payments. If the participant does not consent to Resistance Exercise, the second payment 
will be requested after completion of Recovery Week 4. If participant is unable to complete all 
study visits, the stipends will be prorated to the following: 
 
Visit 
 
Prorated Amount 
SV2 
 
$160.00  
Payment 1 
$1770.00  
PBR Day 1-5 ($50/day) 
 
$250.00  
BR Day 1-9 ($110/day) 
 
$1100.00 
BR Day 10 
 
$160.00 
Post Bed Rest Day 11 
 
$100.00 
Recovery Week 1 
 
$250.00  
Recovery Week 2 
 
$250.00  
Payment #2 
Up to 
$1780.00 
Recovery Week 3 
 
$250.00  
Recovery Week 4 
 
$250.00  

 
 
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Optional Resistance Week 1 
($65/session) 
 
$195.00  
Optional Resistance Week 2 
($65/session) 
 
$195.00  
Optional Resistance Week 3 
($65/session) 
 
$195.00  
Optional Resistance Week 4 
($65/session) 
 
$195.00  
Total for all completed visits 
 
Up to $3,550.00  
Up to 
$3,550.00  
 
Publication Plan 
We attest that the AdventHealth TRI faculty and staff will adhere to POLICY-TRI-ADM-005 
(Access to Clinical Trial Data for Publication Purposes). The goal will be to publish novel and 
interesting findings from this research. Assignment of authorship and the contributions of each 
author will be determined by the International Committee of Medical Journal Editors (ICMJE) 
policy to guide authorship. 
 
 
 
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