Vascular Dysfunction During Physical Inactivity: Role of Oxidative Stress
Phase: NA
Conditions: Aging, Vascular Endothelium, Skeletal Muscle
Sponsor: University of Utah

 
 
Vascular Dysfunction During Physical Inactivity 
 
NCT04872998 
 
Document Type: Study Protocol 
 
Document Date: 01MAY2018 

Protocol Summary 
Version: 1, May 2018 
“Understanding Inactivity: Investigating the Impact of Reduced Activity on Vascular and 
Skeletal Muscle Function” 
 
Principal Investigator 
 
Joel D. Trinity, Ph.D. 
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 4333 
joel.trinity@utah.edu 
 
Sub-Investigator(s) 
 
Micah Drumond, Ph.D. 
520 Wakara Way, SLC UT 84108 
801-585-1310 
micah.drummond@hsc.utah.edu 
 
Paul LaStayo 
520 Wakara Way, SLC UT 84108 
801-581-7419 
paul.lastayo@hsc.utah.edu 
 
Russell S. Richardson, Ph.D. 
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
801-582-1565 ext. 4434 
r.richardson@hsc.utah.edu 
 
Markus Amann, Ph.D. 
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
801-582-1565 ext.4358 
Markus.amann@hsc.utah.edu 
 
Amber Bledsoe, M.D. 
30 N. 1900 E RM 3C444, SLC, UT 84132-2101 
801-581-6393 
Amber.bledsoe@hsc.utah.edu 
 
Jacob Jessop, D.O. 
30 N 1900 E RM 3C444, SLC, UT 84132 
801-581-6393 
Jacob.jessop@hsc.utah.edu 
 
Philip Kithas, M.D. 
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 2657 
philip.kithas@va.gov 
 
 
 

 
Dave Mann, Pharm.D. 
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 1454 
david.mann4@va.gov 
 
David Morgan, M.D. 
30 N 1900 E RM 3C444, SLC, UT 84132-2101 
801-581-6393 
david.morgan@hsc.utah.edu 
 
Van Reese, M.S. 
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 4335 
van.reese@hsc.utah.edu 
 
Mark Supiano, M.D. 
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
(801) 587-9103 
mark.supiano@utah.edu 
 
D. Walter Wray, Ph.D. 
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 1556 dwalter.wray@hsc.utah.edu 
 
Angela V. Bisconti, M.S. 
VA Medical Center Bldg. 2, 500 Foothill Drive, SLC UT 84148 
801-582-1565 x 1918 
u6016131@utah.edu 
 
Ryan Broxterman, Ph.D. 
VA Medical Center Bldg. 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 1951 
ryan.broxterman@utah.edu 
 
Kanokwan Bunsawat, Ph.D.  
VA Medical Center Bldg. 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 1951 
Kanokwan.bunsawat@hsc.utah.edu  
 
James Cerbie, M.S.  
VA Medical Center Bldg. 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 4313 
james.cerbie@utah.edu  
  
 
Vincent Georgescu, M.S.  
VA Medical Center Bldg. 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 4313 
james.cerbie@utah.edu  
 

Jay Hydren, M.S.  
VA Medical Center Bldg. 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 4313 
jay.hydren@utah.edu  
 
Catherine Jarrett, Ph.D. 
VA Medical Center Bldg. 2, 500 Foothill Drive, SLC UT 84148 
801-582-1565 x 4333 
Catherine.Jarrett@utah.edu  
 
 
David Taylor LaSalle, M.S. 
VA Medical Center Bldg. 2, 500 Foothill Drive, SLC, UT 84148 
801-582-1565 ext. 3919 
Taylor.lasalle@utah.edu  
 
Soung Hun Park, M.S.  
VA Medical Center Bldg. 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 3919 
sounghun.park@utah.edu  
 
Lisette Perez, Study Coordinator 
VA Medical Center Bldg. 2, 500 Foothill Drive, SLC, UT 84148 
801-582-1565 x 1918 
lisette.perez@hsc.utah.edu 
 
Ella Dixon, Study Coordinator 
VA Medical Center Bldg. 2, 500 Foothill Drive, SLC, UT 84148 
801-582-1565 x 1495 
ella.dixon@hsc.utah.edu 
 
Katherine Shields, M.S. 
VA Medical Center Bldg. 2, 500 Foothill Drive, SLC UT 84148 
801-582-1565 x 4313 
Katherine.Shields@utah.edu  
 
Taylor Thurston, M.S. 
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
taylor.thurston@umail.utah.edu 
 
Joshua Weavil, M.S. 
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
Josh.weavil@utah.edu 
 
Hsuan-Yu Wan, Ph.D. 
VA Medical Center Bldg. 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext.4333 
hsuan-yu.wan@hsc.utah.edu  
 
Jia Zhao, M.S., Wet Lab  
VA Medical Center Bldg. 2, 500 Foothill Drive, SLC UT 84148 

582-1565 ext.4335 
jia.zhao@hsc.utah.edu  
 
 
Ned Morgan, M.D. 
 
Candice Morrissey, M.D. 
 
Scott Junkins, M.D. 
 
Natalie Silverton, M.D 
 
Nathaniel Birgenheier, M.D.  
 
Daniel Pulsipher, M.D. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

BACKGROUND AND INTRODUCTION 
Prolonged periods of reduced activity are associated with decreased vascular function and muscle 
atrophy1-3.  Physical inactivity due to acute hospitalization is also associated with impaired recovery, 
hospital readmission, and increased mortality.4 Older adults are a particularly vulnerable population as 
functional (vascular and skeletal muscle mitochondrial dysfunction) and structural deficits (loss in muscle 
mass leading to a reduction in strength) are a consequence of the aging process.5-8 The combination of 
inactivity and aging poses an added health threat to these individuals by accelerating the negative impact 
on vascular and skeletal muscle function and dysfunction. The underlying factors leading to vascular and 
skeletal muscle dysfunction are unknown, but have been linked to increases in oxidative stress.9 
Additionally, there is a lack of understanding of how vascular function is impacted by inactivity in humans 
and how these changes are related to skeletal muscle function.  It is our goal to investigate the 
mechanisms that contribute to disuse muscle atrophy and vascular dysfunction in order to diminish their 
negative impact, and preserve vascular and skeletal muscle function across all the lifespan. 
 
The CDC recently stated the average length of a hospital stay is between 4-5 days, with older individuals 
(>65 yrs) staying marginally longer, 5-6 days12.  Injuries or illnesses not connected with hospitalization may 
still lead to periods of inactivity during the recovery process.  As such, it is possible for an individual to 
experience multiple periods of inactivity throughout life.  Studies investigating the negative impact of 
physical inactivity have utilized bed rest, limb immobilization, reduced activity and prolonged sitting2, 3, 10-
12. Our objective is to utilize these models to understand and prevent the associated decrements in 
vascular and skeletal muscle function and preserve physiological function. 
 
Bed rest studies have been used to simulate anti-gravity effects during space flight and bed rest during 
hospitalization13, 14.  Bed rest studies have shown muscle atrophy and strength loss, but are time intensive 
and expensive to administer. A common alternative to the bed rest model has become a single leg 
immobilization model, with either a knee brace or leg cast used to immobilize one leg.  Recent studies 
have shown that short periods of limb immobilization (5-7 days) have caused muscle atrophy with an 
accompanied loss of strength2, 10. Reducing activity by closely monitoring and decreasing daily step counts 
by 75% have also shown small decreases in muscle mass, along with impairments in insulin sensitivity and 
increased inflammatory markers12. Furthermore, investigators have shown that sitting while being 
inactive for 3-6 hours can impair vascular function in the lower limb11. Taken together these collective 
studies show the negative impact of physical inactivity.  
A proposed mechanistic link between periods of inactivity and the ensuing decrements in skeletal muscle 
and vascular function, is an increase of oxidative stress. Oxidative stress is also linked with an age related 
decrease of vascular function.  Non-specific antioxidant supplementations have been shown to restore 
vascular function to levels that are indistinguishable from healthy young adults15-18. Animal studies 
utilizing prolonged skeletal muscle disuse models have shown oxidative stress increases in the inactive 
muscle, with mitochondria-targeted antioxidants restoring function with a reduction of oxidative19-21. We 
propose that oxidative stress is one mechanism that is being triggered by inactivity and may, in part, be 
responsible for the negative vascular and skeletal muscle function due to the insult of physical inactivity.   
One pathway that is responsible for combating oxidative stress in the body is the activation of NF-E2-
related factor 2 (Nrf2). Activation of Nrf2 occurs when it is released from Keap1in the cytosol and 
translocates to the nucleus. In the nucleus, Nrf2 promotes transcription of antioxidant enzymes.  
Protandim and PB125 are Nrf2 agonist supplements which likely activate Nrf2. Protandim has been shown 
to activate Nrf2 and increase antioxidant enzyme expression during an oxidative challenge in human 
coronary aortic endothelial cells22.  We propose the use of non-specific antioxidants, mitochondria-

targeted antioxidants and a Nrf2 stimulator to determine the impact of oxidative stress on physical 
inactivity.    
Exercise training and rehabilitation can be used as an alternative approach to combat the destructive 
effects of oxidative stress on aging and disease. An effective exercise training intervention can improve 
arterial compliance and vascular endothelial function23, 24.  Exercise training has been shown to alter the 
pro- and antioxidant balance resulting in improved endogenous antioxidant defense mechanisms15, 25. 
Moreover, exercise training concomitantly improves musculoskeletal strength and function, 
cardiovascular function, body composition, blood chemistry (decreased triglyceride and cholesterol 
levels), and overall well-being26-28.  NASA scientists developed an integrated resistance and aerobic 
exercise program during a strict 14 day bedrest study. With less than one hour per day performing 
exercise, participants preserved muscle size, strength and aerobic capacity29. We aim to uncover the effect 
of exercise training and rehabilitation during physical inactivity.  
We propose that physical inactivity induces a loss of vascular and skeletal muscle function and structure, 
which may be mechanistically linked to increased oxidative stress.  Through the models and pathways 
described previously, our aim is to utilize non-specific antioxidants, mitochondrial-targeted antioxidants, 
Nrf2 promoter supplements and exercise training and rehabilitation in order to understand and mitigate 
the negative impact of physical inactivity. 
 
OBJECTIVES:  An experimental protocol has been designed to specifically evaluate the 
role of inactivity on functional (vascular and mitochondrial) and structural (muscular) 
outcomes in young and old individuals. 
Specific aim 1: Determine the impact of pre-training on functional (vascular and 
mitochondrial) and structural (muscular) outcomes before and after inactivity. Hypothesis: 
Pre-training (or prehabilitation) will increase the reserve of participants as evidenced by functional 
(endothelial and mitochondrial) and structural (muscular strength and size) improvements prior to 
inactivity.  Following inactivity, a higher level of function will be present in those individuals that 
performed pre-training.  It is expected that improved function will be associated with improved 
redox balance as a result of pre-training-induced increases in endogenous antioxidant capacity. 
Specific aim 2:  Determine the impact of exercise during a stint of inactivity on functional 
(vascular and mitochondrial) and structural (muscular) outcomes. Hypothesis: One to 
twenty days of exercise during a period of inactivity will minimize the decrements in functional 
(endothelial and mitochondrial function) and structural (muscular strength and size) outcomes 
evoked by inactivity.  It is expected that the attenuated decline in function will be associated with 
improved redox balance as a result of exercise induced increases in endogenous antioxidant 
capacity. 
Specific aim 3:  Determine the impact of post-training on the recovery of functional 
(vascular and mitochondrial) and structural (muscular) outcomes following a period of 
inactivity. Hypothesis: Four to twelve weeks of post-training (or rehabilitation) following a period 
of inactivity will recover functional (endothelial and mitochondrial function) and structural 
(muscular strength and size) measures lost during inactivity.  It is expected that the augmented 
recovery compared to baseline will be associated with improved redox balance as a result of 
exercise induced increases in endogenous antioxidant capacity. 

Specific aim 4:  Determine the contribution of oxidative stress in inactivity-induced losses 
in functional (vascular and mitochondrial) and structural (muscular) outcomes.  
Hypothesis:  Administration of antioxidants such as an oral antioxidant cocktail (vitamins C, E, 
alpha-lipoic acid), mitochondrial-targeted antioxidant (MitoQ), or a NrF2 agonist (Protandim 
and/or PB125) during inactivity will minimize the decrements in functional (endothelial and 
mitochondrial function) and structural (muscular strength and size) outcomes evoked by inactivity.  
It is expected that the attenuated decline in function will be associated with improved redox 
balance as a result of free radical scavenging and/or increased endogenous antioxidant capacity.  
 
PARTICIPANT SELECTION CRITERIA: 
Inclusion Criteria: 
• 
Healthy individuals 18 years of age or older 
• 
Individuals who have recently undergone surgery or injury requiring inactivity (limb 
immobilization or bed rest) 
 
Exclusion Criteria:  
• 
Age of less than 18 years 
• 
Individuals unwilling or unable to give consent 
• 
Cardiac abnormalities considered exclusionary by the study physician (e.g., CHF, CAD, 
right-to-left shunt) 
• 
Uncontrolled endocrine or metabolic disease (e.g., hypo/hyperthyroidism, diabetes 
[HbA1c > 6.5%])  
• 
GFR <45 mL/min/1.73m2 or evidence of kidney disease or failure 
• 
Vascular disease or risk factors of peripheral atherosclerosis. (e.g., uncontrolled 
hypertension, obesity, diabetes, hypercholesterolemia > 250 mg/dl, claudication or 
evidence of venous or arterial insufficiency upon palpitation of femoral, popliteal and 
pedal arteries) 
• 
Risk of DVT including family history of thrombophilia, DVT, pulmonary emboli, 
myeloproliferative diseases including polycythemia (Hb>18 g/dL) or thrombocytosis 
(platelets>400x103/mL), and connective tissue diseases (positive lupus anticoagulant), 
hyperhomocystinemia, deficiencies of factor V Leiden, proteins S and C, and 
antithrombine III 
• 
Use of anticoagulant therapy (e.g., Coumadin, heparin) 
• 
Elevated systolic pressure > 180 or a diastolic blood pressure > 110 
• 
Implanted electronic devices (e.g., pacemakers, electronic infusion pumps, stimulators) 
• 
Cancer or history of successfully treated cancer (less than 1 year) other than basal cell 
carcinoma 
• 
Currently on a weight-loss diet or body mass index > 35 kg/m2 
• 
Inability to abstain from smoking for duration of study 
• 
A history of > 20 pack per year smoking 
• 
HIV or hepatitis B or C*- 
• 
Recent anabolic or corticosteroids use (within 3 months) 
• 
Subjects with hemoglobin or hematocrit lower than accepted lab values 
• 
Agitation/aggression disorder (by psychiatric history and exam) 
• 
History of stroke with motor disability 
• 
A recent history (<12 months) of GI bleed 
• 
Depression [>5 on the 15 items Geriatric Depression Scale (GDS)] 
• 
Alcohol or drug abuse 

• 
Liver disease (AST/ALT 2 times above the normal limit, hyperbilirubinemia) 
• 
Respiratory disease (acute upper respiratory infection, history of chronic lung disease with 
resting oxygen saturation <97% on room air) 
• 
Women who are pregnant 
• 
Any other condition or event considered exclusionary by the PI and faculty physician 
 
DESIGN:  The proposed work falls within the scope of a single protocol.  Specifically, this study 
is designed to investigate the impact of inactivity (in the form of bed rest, limb immobilization or 
reduced activity) and oxidative stress on functional and structural outcomes.  All procedures for 
the proposed protocol are strictly research-related. 
Pregnancy testing will be performed on all women of child bearing age and potential.  Any 
pregnant participants will be excluded from the study. 
STUDY PROCEDURES 
An overview of each visit to the laboratory, including chronology of the protocol, is listed 
on the following pages. This is followed by separate headings which provide details for 
participants, instrumentation, and the techniques and measurements identified in the 
protocol.   
 
PRELIMINARY SCREENING AND BASELINE DATA COLLECTION (all participants, 1-2 
hours/day) 
After obtaining informed consent, participants will report to the laboratory (VA Bldg 2, Room 1D21) 
for orientation, completion of medical history and activity questionnaires, and may have a medical 
examination performed by one of the study physicians. A screening questionnaire will be filled out 
that will include questions concerning risks such as: age, family health history, smoking, 
hypertension, hypercholesterolemia, and physical activity and allergies (iodine, latex, drugs, etc). 
A venous blood sample (4 ml) may be drawn from which a Metabolic Panel, Complete Blood 
Count (CBC), and a Lipid Panel will be assessed.  
Next, eligible participants may be asked to complete one or more of the following exercise tests: 
a graded exercise test to volitional exhaustion (bike or treadmill) with or without a mouthpiece to 
determine oxygen consumption, maximal and sub-maximal handgrip test, maximal and sub-
maximal leg extension test, and maximal and sub-maximal plantar flexion test. Sufficient recovery 
will be allowed between tests, and ECG will be assessed at all times. The hand grip, leg extension, 
and plantar flexion graded exercise tests are considered low cardiovascular risk tests due to the 
recruitment of an isolated small muscle mass that results in minor cardiovascular stress. The 
graded whole-body exercise tests to volitional exhaustion (bike, treadmill), when performed in 
participants over the age of 45 will be completed under physician supervision, and the participant 
will be monitored by an ECG throughout the exercise test. Any abnormal ECG responses during 
the exercise test (overt arrhythmia, severe [3 mm] ST segment depression, elevated [1mm] ST 
segment in non-Q wave lead, frequent extra-ventricular systoles) or adverse responses such as 
chest pain, leg pain, or unsteadiness will serve as criteria to stop the test, in accordance with 
American Heart Association guidelines. Questionable findings will be referred to a cardiologist for 
inspection/diagnosis prior to further involvement in the protocol. Additionally cognitive 
assessment such as measuring the participant’s ability to follow basic instruction regarding 
exercise training and physical assessments such as a 6-minute walk test may be performed. 
Additionally, participants may be asked to wear an accelerometer to measure activity levels. 

If the participant is unable to complete the testing listed above additional visits may be scheduled 
to complete the testing.  
Following the exercise testing, participants may be given instruction and equipment for physical 
activity monitoring. 
Due to the nature of the testing performed under this protocol, we will not enroll pregnant women. 
Additionally, women of child-bearing age will be asked to undergo a urine pregnancy test provided 
by the research site to confirm that these female participants are not pregnant. Additionally, these 
participants will be asked not to become pregnant while participating in this protocol. If a female 
participant becomes pregnant she will be asked to inform the PI or other designee immediately 
and will be withdrawn from any part of this protocol.   
EXPERIMENTAL PROTOCOL: 
Prior to reporting to the laboratory participants will be enrolled in one of the following modes of 
inactivity:   
• 
Bed Rest (1 - 10 days) performed at the University of Utah Center for Clinical and 
Translational Science (CCTS) 
• 
Limb Immobilization (1 - 21 days) 
• 
Reduced Activity (3 hours - 14 days) 
* Detail of each inactivity protocol is described below 
 
Participants may perform exercise training before (1-6 weeks), during, or after (4-12 weeks) 
inactivity (see Participant Instrumentation for more detailed description of exercise protocols).  
Participants may undergo a period of dietary supplementation (1-20 days) by consuming 
Protandim (PRO, 675mg/day), PB125 (100-200mg/day) an antioxidant cocktail consisting of 
vitamins C (500mg), E (200IU) and alpha lipoic acid (300mg), or the mitochondrial targeted 
antioxidant Mito-Q (40-80mg) before, during, or after inactivity for 1-20 days.   
Participants may report to the following laboratories (Utah Vascular Research Lab (UVRL) located 
at the VA Medical Center Bldg. 2, the CCTS located in Research Park at 421 Wakara Way, Rm 
360 or the University of Utah Hospital in the Neuro Acute Care Center, the Utah Center for 
Advanced Imaging Research (UCAIR) located at 729 Arapeen Dr., or the Skeletal Muscle 
Exercise Research Facility (SMERF) located in the Department of Physical Therapy at 520 
Wakara Way) in a fasted state before, during, and after the inactivity protocols listed above. During 
these the following tests/evaluations may be performed: 
• 
Blood collection from an antecubital vein 
• 
Muscle biopsy from the gastrocnemius, soleus, or vastus lateralis.   
• 
Flow mediated dilation (FMD) to assess vascular function 
• 
Passive leg movement (PLM) to assess vascular function 
• 
Oral Glucose Tolerance Test (OGTT) and continuous glucose monitoring 
• 
Exercise testing on a Humac Norm Isokinetic Extremity System.   
• 
Body composition assessed by dual x-ray absorptiometry (DEXA) scanner, magnetic 
resonance imaging (MRI), or D3 creatine.   
• 
Assessment of oxygenated blood using Near Infrared Spectroscopy (NIRS) 

• 
Catheters may be placed in the leg (common femoral artery, dorsalis pedis artery, 
posterior tibial artery, or femoral vein) or arm (brachial artery, radial artery, or antecubital 
vein) by a licensed physician using standard sterile technique.  
o Following the catheterization process endothelial cells may be collected using a 
sterile J-wire (see Participant Instrumentation for more detailed description of this 
procedure) by an approved member of the investigative team.  
o Resting measurements of arterial blood pressure, heart rate, and limb blood flow 
(ultrasound Doppler) may be taken, and resting blood samples (arterial and venous) 
may be collected.  
• 
KE, plantar flexion, or handgrip exercise may be performed at several exercise intensities 
including, but not limited to, 25, 50, and 75% maximal work rate (WRmax) or maximal 
voluntary contraction (MVC) as determined during preliminary testing. Each exercise 
intensity may be continued for up to 5 min to achieve steady-state pulmonary oxygen 
consumption. During each work bout the following sequence of events may be conducted:  
1) sampling of femoral arterial and venous blood for the direct measurement of free 
radicals and blood gases,  
2) continuous measurement of central hemodynamics (heart rate, stroke volume, 
cardiac output) and arterial and venous pressures (indwelling catheter pressure), 
and  
3) leg or arm blood flow via ultrasound Doppler. 
 
 
Inactivity Protocols:  
 
1) Bed Rest: After completion of pre-inactivity assessments, participants will adhere to bed rest 
lasting from 1 to 10 consecutive days. This bed rest model was designed to mimic a traditional 
inpatient hospital stay and reflect the level of muscle unloading, rest and recuperation that occurs 
following an acute illness, injury or infection.30, 31 During bed rest participants will spend a majority 
of their time in bed and will be allowed to adjust the hospital bed head height for reading, eating 

and watching television but otherwise will be instructed to lay the bed flat for sleeping. Bathing 
and hygiene activities will be performed at the sink in a wheel chair. Subjects will also access the 
bathroom using a wheel chair. Adherence to bed rest will be monitored by nursing staff 24 hours 
a day.  
2) Limb Immobilization: After completion of the pre-inactivity assessments, participants will 
adhere to unilateral leg immobilization which will be accomplished by a removable knee brace set 
at 60⁰ flexion (for 1 - 21 days). Crutch assisted locomotion will be utilized to minimize weight 
bearing of the immobilized leg. Unilateral limb immobilization for 14 days in humans provides an 
effective and feasible model to evoke significant structural and functional losses in response to 
unloading resulting in a  ~20% reduction in muscle strength, 10% reduction in exercise capacity, 
5-6% loss of leg lean masses, and 5-10% decrease in fiber cross sectional area 32-35. 
3) Reduced Activity: After completion of the pre-inactivity assessments, subjects will adhere to 
either short term (3 to 6 hours of sitting) or extended (1-14-days) of reduced physical activity at 
their home/work as has been conducted before.12, 36 During the short term protocol subjects will 
remain in the seated protocol with minimal to no movement of the lower extremities as has been 
described previously.3, 11, 37, 38 During reduced activity, the goal will be for the participant to drop 
their physical activity levels to 75% of their normal activity levels as determined by a monitor that 
the subject will be able to see and record. We will inform the participant that their normal diet 
should remain the same. A second step monitor will be taped to their leg (waterproof) for the 
investigators to assess the participants' compliance to the assigned step/day guidelines. For the 
extended protocols lean mass, strength, power, and physical function measurements will be re-
assessed on a later day following the reduced activity study. 
  
PARTICIPANT INSTRUMENTATION (alphabetical order) 
Antioxidant and blood measures: Ascorbic acid will be measured in the plasma by the vitamin 
C assay kit (CosmoBio, Carlsbad, CA). Ferric Reducing Ability of Plasma will be determined 
according the spectrophotometric technique previously described 39. The FRAP method uses the 
ability of antioxidants to reduce a FE3+-TPTZ complex to its blue colored Fe2+ form. GSH, GSSG 
and Total Glutathione will be measured using BioVision Glutathione Assay Kit (#K264-100) (San 
Francisco, CA). Lipid hydroperoxides will be assessed by the lipid hydroperoxide (LPO) assay 
kit (Caymen, Ann Arbor, MI). Nitrite/Nitrate levels will be measured in the plasma using a 
standard fluorometric assay kit (Caymen, AnnArbor, MI). 8-isoprostane, a marker of lipid 
perodixation and oxidative stress, will be analyzed in the plasma using Cell Biolabs 8-iso-PGF2α 
kit (#STA-337) (San Diego, CA).  
Antioxidant Cocktail: An antioxidant cocktail consisting of vitamins C (500mg), E (200IU) and 
alpha lipoic acid (300mg) will be consumed daily for 1-20 days. The PI’s and Co-I’s have 
previously utilized antioxidant cocktail supplementation in studies of both aging and disease when 
testing the ability of antioxidant supplementation to prevent free radical-mediated damage 40-45. 
Blood Draw: Blood draws will be performed by Dr. Trinity or one of the other trained investigators 
in phlebotomy technique in our group or by CCTS nursing staff. In the rare case that this 
procedure is particularly difficult, as this is a standard procedure performed in our laboratory, the 
participant will be escorted to the VA Medical Center's blood laboratory in an adjacent building 
where full-time phlebotomists will perform the blood draw.  
Body Composition: Dual X-ray Absorptiometry (DEXA) will be used to measure body 
composition (% body fat, % Lean Tissue, Bone Density) non-invasively. This test will be 

performed while lying on a padded bed while the low-level x-ray probe passes over the 
participant’s body (0.0004 cGy radiation exposure associated with the DEXA). Body composition, 
specifically total body muscle mass, may be determined by the creatine (methyl-d3) dilution 
method 46. This methods involves consumption of 30mg enriched creatine, provided in a pill 
capsule, and subsequent urine analysis of deuterated and unlabeled creatine by liquid 
chromatography mass spectrometry.  
Catheterization: At each catheter insertion site, local anesthesia with lidocaine will be applied 
(1% lidocaine; 1.33mg/kg assuming 75 kg body weight, with an upper limit of 10 ml). Sterile 
technique will be applied to minimize risk of infection. 1.0 mg of atropine will be available in case 
of sudden vagal reactions. The leg (femoral artery and vein, 18G Cook Catheter or equivalent) 
will be cannulated. A qualified and privileged physician will be responsible for the placement and 
removal of these catheters. Following catheter removal, wounds will be dressed and the 
participants educated as to proper care. Participants will not be released until hemostasis is 
clearly present. Follow-up contact with the participant will continue over the following two days. It 
should be noted that the Drs. Trinity, Richardson, Supiano and Wray have significant experience 
with catheter-related studies 47-56, having coordinated the use and insertion of these exact 
catheters hundreds of times in the last twenty years without incidence of bleeding, infection or 
tissue damage. In fact, several participants have performed multiple studies with our group, 
indicating the benign nature and positive outcome of the procedures. 
Central hemodynamics: Heart rate, stroke volume, cardiac output, and mean arterial pressure 
will be determined non-invasively with a finometer (Finapres Medical Systems, Amsterdam, The 
Netherlands) positioned at heart level. The finometer uses beat by beat arterial pressure 
waveform analysis as assessed by photoplethysmography using the Modelflow method 
(Beatscope, version 1.1; Finapres Medical Systems), which in combination with heart rate has 
been documented to accurately estimate cardiac output during a variety of experimental protocols 
57, 58. This technique requires a blood pressure cuff be placed on the upper arm (biceps) and on 
the finger of the participant. Mean arterial blood pressure will also be measured by an automatic 
sphygmometer (Tango+, Suntech, North Carolina) at rest and during exercise.  
Continuous Glucose Monitoring: A commercially available continuous glucose monitor will be 
placed on the participant by CCTS nursing staff or team physician. This device will record 24 hour 
glucose responses (including in response to meal intake) over the inactivity period.  
Diet Stabilization during Bed Rest: Metabolic variability will be reduced by standardizing the diet 
throughout the hospital stay. Meals will be prepared by the University of Utah CCTS Metabolic 
Kitchen (dietician) using the Harris-Benedict equation to estimate daily energy requirements, 
including adjustments for inactivity, and based on the information from a dietician-patient dietary 
interview, and the nutrient distribution recommended by the American Dietetic Association. The 
absolute protein content of each meal will be calculated using the pre-bed rest body weight. 
Macronutrient intake will be evenly distributed between three meals, and uneaten food items will 
be recorded. Dietary data will be analyzed using The Food Processor software program (Salem, 
Oregon). 
 
Humac Norm Isokinetic Extremity System:  Isokinetic and isometric strength will be assessed 
in both legs through maximal voluntary contraction effort developed by the knee extensors/flexor 
(quadriceps/hamstrings) and plantar/dorsi flexor muscles on a CSMI Humac Norm dynamometer.  
 
Electron paramagnetic resonance spectroscopy (EPRS) Ex-vivo spin-trapping with PBN (190 
mM/L in 0.9% NaCl) will be utilized for the downstream detection of lipid-derived radical species 
in blood, as previously described 59-61. We will use the spin-trap concentration of 3:1 ratio of whole 

blood-trap that has proved optimal in our previous studies. We will also use a series of ferrous 
iron-dithiocarbamate ligand spin-trap complexes to directly assess NO concentration in blood. 
Briefly, spin-trap adducts will be extracted from serum (or solute in the case of tissue) into toluene, 
vacuum degassed for 2 cycles and analyzed at 21°C using an EMX X-band EPR spectrometer 
fitted with an ER TM110 cavity (Bruker, Karlsruhe, Germany). Spectrometer conditions will be as 
follows: 20mW experimentally validated non-saturating incident microwave power, 0.5 G 
modulation, 1 x 105 receiver gain, 82 msec time constant, 3450G magnetic field centre and ± 50G 
scan width, for 15 incremental scans. EPR spectral parameters will be obtained using 
commercially available software (Bruker Win EPR System, Version 2.11) and filtered identically. 
The average spectral peak-to-trough line height will be considered a measure.  
 
Endothelial Cell (EC) collection and protein expression: The procedures for the collection of 
EC and measurement of endothelial protein expression have been previously described 62. Briefly, 
following the placement of an arterial (placed by a physician) or venous (placed by an approved 
member of the investigative team) 1 to 5 sterile J-wires are advanced through the catheter into 
the artery and/or vein (~4 cm beyond the tip of the catheter) and retracted through an 18-gauge 
catheter by an approved member of the investigative team, then transferred to a dissociation 
buffer solution, where ECs are recovered by washing and centrifugation. Collection of ECs may 
be performed both before and after an acute intervention of exercise, inactivity, or dietary 
supplementation. EC protein expression will be determined according the method described by 
Donato et al 2007 62. Following EC collection, collected cells will be fixed with 3.7% formaldehyde 
and plated on poly-L-lysine coated slides (Sigma Chemical, St Louis, Mo). For 
immunofluorescence staining ECs will be rehydrated with PBS and nonspecific binding sites 
blocked with 5% donkey serum (Jackson Immunoresearch, West Grove, Pa). Afterward cells will 
be incubated with monoclonal antibodies for one of the following: nitrotyrosine (Abcam; 
Cambridge, Mass), xanthine oxidase (US Biological; Swampscott, Mass), NAD(P)H oxidase-
p47phox (Abcam; Cambridge, Mass), CuZn SOD (Upstate; Lake Placid, NY), Mn SOD (Research 
Diagnostics; Concord, Mass), catalase (Abcam; Cambridge, Mass) or NF-KB p65 (Novus; 
Littleton, Colo). Cells will next be incubated with CY3-conjugated secondary antibodies (Research 
Diagnostics; Concord, Mass). For analysis, slides will be viewed using a fluorescence microscope 
(Eclipse 600, Nikon, Melville, NY) and EC images will be digitally captured by a Photometrics 
CoolSNAPfx digital camera (Roper Scientific, Inc., Tucson, Ariz). ECs will be documented by cell 
staining of von Willebrand factor and nuclear integrity will be confirmed using DAPI (4',6'-
diamidino-2-phenylindole hydrochloride) staining. Once endothelial cells with intact nuclei are 
identified, they will be analyzed using Metamorph Software (Universal Imaging Corp, 
Downingtown, Pa) to quantify the intensity of CY3 staining (i.e. average pixel intensity). Values 
will be reported as ratios of EC protein expression/human umbilical vein EC (HUVEC); this 
minimizes the possible confounding effects of differences in intensity of staining among different 
staining sessions. 
 
Exercise Prescription during Limb Immobilization:  Total time for each exercise session 
includes allotted time for warm-up and cool-down before and after resistance and aerobic interval 
exercise training sessions. 

 
Pre and post inactivity exercise training (i.e.; prehabilitation and rehabilitation):  The  
prehabilitation and rehabilitation interventions mimic the cardiac rehabilitation program currently 
used by the VA SLC and University of Utah Cardiac Rehabilitation centers with modifications 
developed in conjunction with Dr. Paul LaStayo (Co-Investigator, Physical Therapy) to optimize 
improvements in aerobic capacity and skeletal muscle strength. The prehabilitation and 
rehabilitation interventions will include the following: 
A. Initial Orientation: 
1) Preliminary questionnaires to assess diet, current physical activity, and psychosocial 
health (SF-12-Health Survey, PHQ-9 -Patient Health Questionnaire to assess depression, 
DASI-Duke Activity Status Index Score, and DRA-Dietary Risk Assessment) 
2) Interview with a registered nurse to review medications and past medical history. 
3) Cardiopulmonary physical therapy examination to assess readiness for exercise. 
4) Anthropometric measurements – waist circumference, height, weight, body mass index 
calculation (BMI) 

5) Cycle ergometry test to assess exercise capacity and 10 repetition maximum testing to 
determine appropriate workloads for resistance exercises. 
6) Initial exercise prescription will be based on results of the cycle ergometry and 10 
repetition maximum tests.The personalized PREHAB exercise prescription plan is 
described below (See 3. Exercise Prescription, Performance, and Progression). 
 
B. Pre-exercise assessment: 
1) Weight is obtained from subject. 
2) Resting blood pressure and heart rate are obtained. 
3) Subjects’ telemetry is evaluated before, during, and after exercise. 
4) Proper warm-up exercises are taught, demonstrated, and performed. 
 
C. Exercise Prescription, Performance, and Progression: 
1) Subjects will perform PREHAB or REHAB 3 times per week for the duration of the 
intervention. 
2) The prescribed exercise is based on the individual’s baseline aerobic capacity and 
strength. 
3) Evaluations of aerobic capacity and strength will be assessed at 2 week increments in 
order to adjust exercise intensities and maintain an adequate training stimulus. The 
exercise intensity for aerobic exercise will be set at predetermined work rates (watts) 
known to elicit a given % of VO2max. The work rate verse VO2 relationship will be 
determined during baseline VO2max and ventilatory threshold testing. As aerobic capacity 
increases absolute work rates will be progressively increased. The intensity for resistance 
exercise will be based on 10 repetition maximum. During the final set of each resistance 
exercise subjects will be instructed to continue until volitional exhaustion. If the subject is 
able to perform more than the prescribed number of repetitions, the load will be increased 
accordingly for the next session. If the subject is not able to complete the prescribed 
number of repetitions the load will be maintained or decreased accordingly for the next 
session. Abbreviations: DB (dumbbell). 

 
D. Post-Exercise Assessment: 
1) Proper stretching exercises are taught, demonstrated, and performed. 
2) Subject rests for five minutes and post-exercise blood pressure and heart rate are 
obtained. 
 
E. Documentation of Exercise Sessions: 
1) All data collected during an exercise session is written on an intake sheet. 
2) All data is then entered into EPIC. 
 
F. Final Evaluation: 
At the completion of the prehabilitation and rehabilitation program, the subject will receive an 
exit interview to review the following: 
1) Preliminary and final tests are given to subject to evaluate outcomes. 
2) Subjects’ current exercise prescription 
3) Subjects’ anthropometric measurements 
4) Summary of the outcomes obtained by the subject through the program. 
5) Note is forwarded to referring physician and Medical Director (Dr. Supiano). 
   
Exercise Testing: Passive leg movement will be performed by a member of the research team 
moving the participant’s leg through a normal range of motion (90°- 180°).  
Single contraction exercise protocols will be performed with either the upper (handgrip) or lower 
(knee extension) extremity. A single maximal voluntary contraction (MVC) will be established for 
each participant using a handgrip or knee extension dynamometer with an analog output. This 
MVC value will be used to calculate 10%, 20%, 30%, 40% and 50% of MVC for the single 
contraction. Following a 2 min baseline period the participant will perform the rapid (< 1 sec) single 
contraction and central and peripheral hemodynamics will be assessed for 1 min post contraction.  
Active leg exercise will be performed using a custom-built knee-extensor ergometer with the 
participant moving the leg between 90°- 180° at a rate of approximately 60 rpm.  
Plantar flexion exercises will be performed using an ergometer specifically designed to isolate the 
gastrocnemius and the soleus complex at a rate of approximately 60 rpm. 
Handgrip exercise will be performed with the participant laying supine on the laboratory bed with 
handgrip dynamometer in their hand. The arm will be extended perpendicular to the participant’s 
torso and supported by a height-adjustable table. Doppler ultrasound will be performed by a 
trained sonographer in order to obtain brachial artery blood flow measurements. Exercise will be 
performed for 4 stages each lasting 3 - 5 minutes and separated by at least 1 min of rest. Intensity 
will range from 10 to 60% of the participant’s maximal handgrip strength. During exercise ECG 
and blood pressure will be continuously monitored. Arm exercise will involve rhythmic handgrip 
using a computer-interfaced dynamometer.   
The graded exercise test will be performed on a cycle ergometer or treadmill using a conventional 
ramp test to exhaustion. Work rate/exercise intensity will be increased by an appropriately tailored 
level from 2.5-25 watts in each step, depending upon capabilities. Pulmonary ventilation and gas 
exchange will be determined continuously using a low-resistance two-way breathing valve (Hans-
Rudolph 2700, dead space 90 ml) and a pneumotachometer (Fleisch no. 3) on the expiratory 
side. Signals will be assimilated by a PC and O2 and CO2 analysis will be performed in real time 
(Parvo Medics, UT). All of this equipment is currently in place and has been used routinely in our 
laboratory with both healthy volunteers and patients. Throughout all our exercise testing, safety 

is ensured through careful echocardiogram (ECG) monitoring, availability of a defibrillator, an 
oxygen source, emergency drugs, and attendance of qualified personnel. 
Flow Mediated Dilation (FMD): After 15-20 minutes in the supine position a blood pressure cuff 
will be placed on the leg or forearm. Ultrasound images of the artery and Doppler waveforms will 
be obtained prior to cuff inflation (baseline). The cuff will then be inflated to 250mmHg for 5 
minutes. Following the cuff release, ultrasound images of the popliteal artery and Doppler 
waveforms will be recorded continuously for 2 minutes. During this time, arterial images during 
diastole will be captured by image analysis software (Medical Imaging Applications). All recorded 
ultrasound images and velocity spectra segments will be saved to the GE Logiq 7 hard drive for 
off-line image and waveform analysis. The time averaged, and intensity-weighted mean blood 
velocity (Vmean) will be averaged across 5 second intervals for the entire 2 minute clip following 
cuff release. In addition, edge detecting software will be utilized to measure arterial diameter 
frame by frame, which will also be averaged into 5 second intervals. Summed shear rate 
throughout the 2 minutes will be calculated based on vessel diameter and blood velocity. The 
maximum change in vessel diameter, relative to baseline, will be determined and expressed as 
%FMD and %FMD/summed shear rate. A greater FMD response is an indicator of healthier 
vessels. A typical response for a young healthy participants would be 8 - 12% or greater dilation, 
whereas a poor response would be <5%. In addition, it is also important to be able to express the 
change in diameter relative to shear stress, as the shear stress is the stimulus for the NO-induced 
dilation. Again, greater FMD/shear values are indicative of healthier vessels. 
Limb blood flow:  Blood flow will be measured with an ultrasound Doppler (Logiq 7, GE) 
equipped with linear array mechanical sector transducers operating at an imaging frequency of 
10-14 MHz. Vessel diameter will be determined at a perpendicular angle along the central axis of 
the scanned area, where the best spatial resolution can be achieved. The blood velocity profile 
will be obtained using the same transducers with Doppler frequency of 4.0-5.0 MHz, operated at 
high-pulsed repetition frequency mode (2-25 kHz) with a depth of 1.5-3.5cm. Special care will be 
taken to avoid aliasing, to ensure that probe position is stable, the insonation angle does not vary, 
and that the sample volume is positioned in the center of the vessel and adjusted to cover the 
width of the diameter and the blood velocity distribution. Blood velocity measurements will be 
obtained with the probe at an appropriate angle to maintain an insonation angle of 60º or less and 
the sample volume centered. Using arterial diameter and mean velocity (Vmean), blood flow will 
be calculated as: Blood Flow (mL/min) = Vmean · π · (Vessel Diameter/2)2 · 60. 
MitoQ:  A mitochondrial targeted antioxidant supplement containing the antioxidant quinone 
moiety covalently attached to a lipophilic triphenylphosphonium cation (TPP).  The TPP moiety 
on MitoQ enables its accumulation within mitochondria driven by the negative membrane 
potential.  Once within mitochondria, nearly all the accumulated MitoQ is adsorbed to the matrix 
surface of the inner membrane where it is continually recycled to the active quinol antioxidant 
form by complex II in the respiratory chain.63  Participants will consume 40-80mg/day for 1-20 
days as previously described.64, 65 
Muscle biopsies: All biopsies will be taken from either the gastrocnemius approximately 3.5 cm 
deep, anteromedial aspect of gastrocnemius muscle belly 10 cm distal to the tibial tuberosity or 
the vastus lateralis approximately 3.5 cm deep, 15 cm proximal to the knee and slightly distal to 
the ventral mid-line of the muscle.  The 5 mm diameter biopsy needle (Bergstrom) is attached to 
sterile tubing and a syringe to apply a negative pressure to assist in the muscle sample collection 
66. Lidocaine (2%) will be used as local anesthetic. The Principal Investigator has been involved 
in over 100 of these procedures with no negative outcomes. Additionally, in support of this 
technique, a series of subjects recently underwent 5 biopsies each over a period of time, 
illustrating that the biopsy experience is acceptable to the subjects.  The muscle samples from 

each biopsy will be immediately frozen in liquid nitrogen and stored at -80oC for future microscopy, 
EPR spectroscopy, molecular and biochemical assays or immersion-fixed in gluteraldehyde for 
EM analysis. With our current technique and experience, utilizing 2-3 passes of the needle and 
double cut, we are able to attain as much as 300-400 mg of muscle which will permit the 
performance of all proposed analyses.  
Oral Glucose Tolerance Tests (OGTT): This is a standard clinical procedure to determine the 
blood glucose and insulin responses to a standardized glucose challenge. This takes 
approximately 2 hours. Glucola, a sugary beverage containing 75g of glucose, will be 
administered orally over 2 to 5 minutes. Blood samples from an intravenous catheter will be 
determined before and 30, 60, 90, and 120 minutes after Glucola consumption. Approximately 5 
mls of blood will be collected during the OGTT.    
Passive Leg Movement (PLM): Participants are moved into an upright sitting position for ∼10 
min before the start of the data collection and remain in this position throughout the entire protocol. 
The protocol consists of 60-s of resting baseline data acquisition followed by a 2-min bout of 
passive leg extension. PLM is achieved by a member of the research team moving the 
participant's lower leg through a range of motion, defined by 90° and 180° knee joint angles, at a 
rate of 1 Hz. Throughout the protocol, the non-moving leg remains fully extended and supported. 
Real-time feedback to the investigator is provided by a metronome to maintain the cadence. 
Before commencing, and throughout the protocol, participants are encouraged to remain passive 
and resist any urge to assist with leg movement. To avoid a startle reflex and active resistance to 
the PLM, participants are made aware that the assessment will start in the next minute, but to 
minimize the chance of an anticipatory response, they are not informed of exactly when 
movement will begin 122.  There are no known risks of this procedure.  
PB125:  The product PB125 (Pathways Bioscience, Aurora, CO) to be used in this study contains 
extracts derived from three botanical sources: Rosmarinus officinalis, standardized to carnosol 
content of 6%, 68mg of extract; Withania somnifera (aka Ashwagandha), standardized to 
withaferin A content of 1%, 23mg of extract; and Sophora Japonica, standardized to luteolin 
content of 98%, 9mg of extract, to create a total daily dosage of 100mg of combined extracts 
administered in a single capsule administered up to twice daily (i.e. 100-200mg/day). Other 
inactive ingredients consist of rice flour and hydroxypropyl cellulose (vegetarian capsule).  PB125 
meets or exceed GMP quality standards.  The product does not contain gelatin, dairy, yeast, 
wheat, or gluten. 
Physical Activity Assessment:  Physical activity of the participant may be assessed by 
accelerometery (i.e. activity monitor) and/or physical activity questionnaire.  With accelerometry, 
participants will be asked to wear a small activity tracker attached to a belt fastened around their 
arm, leg, or waist for approximately 7 days.  These activity trackers (Actigraph, Actilife, USA) 
simply record the number of steps a person takes each day, and the amount of movement a 
person makes each day.  Participants may also be asked to complete a physical activity 
questionnaire (International Physical Activity Questionnaire, IPAQ) or IPAQ for Elderly123.  These 
short questionnaires simply assess how many minutes per day an individual performs physical 
activity. 
Protandim:  A nutritional supplement comprised of five plant extracts (milk thistle, bacopa, 
ashwagandha root, turmeric, green tea) that activates the Nuclear factor (erythroid-derived 2)-like 
2, (called Nrf2) transcription factor pathway that is integral to several antioxidant enzymes, 
including γ-glutamyl cysteine synthase (an enzyme that catalyzes the committed step in 
glutathione synthesis).67 Nuclear factor (erythroid-derived 2)-like 2 is a basic leucine zipper protein 
transcription factor that regulates the expression of antioxidant proteins that protect against 

oxidative damage triggered by injury and inflammation.  Participants will consume 675mg/day for 
1-20 days as previously described.68 
DATA SAFETY & MONITORING 
In the interest of participant safety and to ensure compliance with IRB and FDA regulations, we 
propose the implementation of a data safety and monitoring group. Four Utah Vascular Research 
Laboratory faculty members (Drs. Trinity, Richardson, Wray, and Amann), an internal sub-
investigator (Mark Supiano, M.D.), four members external to the UVRL: Dr. Alan Light and Micah 
Drummond, Ph.D., Dorothea Rosenberger, M.D., Jacob Jessop, M.D. and Lisette Perez and 
Shelby Love, coordinators for the DSMB will make up the monitoring group, all of whom are 
affiliated with the University and the SLC VA Geriatric Research Education and Clinical Center. 
These members will meet on a quarterly basis, and additionally will convene in response to any 
adverse events or unanticipated problems associated with the proposed studies.  
All participants participate on a strictly voluntary basis, and thus any protocol may be stopped at 
any time and for any reason at the participant’s request. Additional criteria to discontinue a 
protocol will be any evidence that the participant’s health is at risk, as identified by a member of 
the investigative team. Participant safety is at the forefront of all studies, and for this reason all 
investigators are trained to identify overt (i.e. abnormal ECG patterns, acute change in blood 
pressure, etc.) and more subtle (i.e. skin pallor or redness, changes in participant demeanor, etc.) 
signs that a participant may be adversely affected by the experimental interventions.  Adverse 
events and unanticipated problems are broadly defined as any instance of a medical complication 
arising from catheter insertion (i.e. excessive bleeding and swelling at insertion site) or drug 
infusion (i.e. acute hypertension, bradycardia, etc.) as listed under the “potential risks” section 
below. In the instance of an adverse event, the protocol will be discontinued and the study will be 
temporarily suspended while DS&M members will review the cause of the event. When 
appropriate, unanticipated adverse events will be reported within 10 working days, in accordance 
with the University of Utah IRB Reporting Policy and to any other pertinent regulatory body(ies).  
 
POTENTIAL RISKS  
Due to the nature of the testing performed under this protocol, we will not enroll pregnant women. 
Additionally, women of child-bearing age will be asked to undergo a urine pregnancy test provided 
by the research site to confirm that these female participants are not pregnant. Additionally, these 
participants will be asked not to become pregnant while participating in this protocol. If a female 
participant becomes pregnant she will be asked to inform the PI or other designee immediately 
and will be withdrawn from any part of this protocol.   
Bed Rest Risks: Bed rest will be performed at the University of Utah CCTS with around-the-clock 
medical supervision by trained medical staff. To ensure safety and comfort during the 5 days of 
bed rest we will follow our established guidelines presented below: 
• A slight bed-back elevation will be permitted and subjects may raise their shoulders with two 
pillows.  
• Subjects will be encouraged to change position periodically to alleviate positional 
discomfort and to eat. 
• Serial compression devices and compression stockings will be worn to decrease DVT risk. 
• Physical therapy will be provided daily to reflect standard of care for bedridden individuals. 
It will include passive range of motion to lower extremity major joints in all planes (~10 
repetitions/plane) 

• Bathing and hygiene activities will be performed during bed rest.   
• A bedside wheelchair will be provided to transport to the bathroom for urine excretion and 
bowel movements. 
• Daily safety blood draws (D-dimer, complete blood count with platelet count and auto-
differential, partial thromboplastin time, prothombin time). 
• 24h nursing supervision and care and medical oversight by team physician (Co-I: Mark 
Supiano, MD) 
 
Following the bed rest period, participants will be immediately evaluated by a physical therapist 
and CCTS nursing staff to deem if the patient is safe to be discharged from the hospital research 
unit (CCTS). We have observed that after 5-days of bed rest, healthy older adults experience 
losses in muscle mass and strength but do not exhibit functional or cognitive impairments and are 
well enough to return home without inpatient exercise rehabilitation. Patients may return to the 
CCTS the following day for a MRI scan to determine the effects of bed rest on leg lean mass and 
afterwards visit the SMERF laboratory for knee extensor leg strength assessment as outlined in 
the experimental timeline. 
 
Limb Immobilization Risks: Limb immobilization is an established model of inactivity resulting in 
significant muscle atrophy (~4-5%) and losses in muscle strength (~20%) and exercise capacity 
(~10%). During limb immobilization participants will remain ambulatory by use of crutches. 
Participants will be instructed on proper use of crutches by licensed Physical Therapist in an effort 
to minimize risks of falling and injury. During limb immobilization there is an increased risk of deep 
vein thrombosis. To minimize this risk periodic blood draws (every 3rd day) during limb 
immobilization will be performed and assessed for D-dimer, complete blood count with platelet 
count and auto-differential, partial thromboplastin time, prothombin time. Additionally, participants 
will be instructed to perform unweighted hip, knee, and ankle contractions at least twice daily to 
activate the muscle pump and further minimize the risk of DVT. Approximately 10 contractions at 
each joint will be performed. If advised by the team physician compression stockings may also be 
worn during limb immobilization.  
Myocardial ischemia, infarction, arrhythmia, and death during exercise. These risks will be 
minimized by the initial pre-screening and non-invasive testing of each participant. If problems 
are found the participant will be excluded and referred to his/her physician. The responses to 
absolute and relative indications for the termination of an exercise test are variable depending 
upon the finding. However, an incident of ischemia presented as chest pain, shortness of breath, 
acute hypotension (<80/40 mmHg), or abnormal ECG findings (>2-3 mm ST depression or 
appearance of S3 or S4 heart sounds) would result in the immediate termination of exercise. The 
participant would be placed in a supine position, administered oxygen and assessed by the on-
site physician and research staff. If the condition did not improve or worsened 911 would be called. 
In all cases this occurrence would be documented and the participant would ultimately be referred 
to their physician. Cardiac function will be monitored during all exercise studies with an ECG 
monitor. 
Biopsy risks: Using sterile technique and a minimal skin incision (about 1/8”) over the anterolateral 
mid-thigh, the standard needle aspiration method for muscle biopsy has not produced bleeding 
nor infectious complications to date. There is the possibility of a small scar the size of the incision 
(1/8”).  No subject is released until hemostasis has been secured, and the skin site dressed.  
Subjects are always carefully instructed in the routine prevention of infection and bleeding of 
biopsy sites. As lidocaine is used as the anesthetic there is the risk of toxicity, however the low 
concentration 0.5% and the use of less than 10-15 ml greatly reduces this concern. Though 
multiple biopsy procedures create additional sites which may be prone to infection, as outlined 

above the technique employed minimizes this risk. Nonetheless, on protocols where multiple 
biopsies are performed, special care will be taken with the sterile dressing and bandaging to 
further minimize risk of infection. Further, the small quantity required for each sample (300-400 
mg) is such that attaining multiple biopsies in one visit is not viewed as presenting a compound 
risk in terms of muscle damage and healing.   
Additionally, nerve injury including paresthesia or motor deficits could occur that could be 
permanent, and chronic muscle atrophy or possibly permanent pain could occur at the biopsy 
site. If an infection occurs post biopsy, antibiotics or even surgery to repair infection damage may 
be necessary.  
Catheter-related risks. There is also the possibility of fainting, dizziness, and possible pain and 
bruising as a result of catheter insertion. There is a risk of infection, swelling, and discomfort at 
the insertion sites, and a risk that bleeding may occur after catheters removal. A clot or excessive 
bleeding at the puncture site could result in a partial blockage of the blood flow to the leg, which 
in extreme cases could lead to loss of the limb. Based on the fact that two arteries supply the 
hand versus only one artery supplies the leg, femoral arterial catheterization bears a greater risk 
to the participants' health. Accordingly, the participants will be allowed to select the site of the 
arterial catheterization, the choices are either femoral or radial artery. These risks are minimized 
by careful patient selection, catheter insertion by a physician with experience using proper 
equipment and sterile methods, a continuous infusion of fluids thorough the catheters, adequate 
pressure for >30 minutes following catheter removal, and appropriate patient education and 
follow-up. The PI and several CO-I’s named herein have significant cumulative experience in this 
areas, having been involved in several hundred successful catheter-based studies with no 
instances of bleeding, infection, femoral vein thrombosis or soft tissue damage as a result of the 
procedures proposed. 
Pregnancy risks:  Due to the fact that little is known about how these procedures will affect a fetus, 
we cannot allow pregnant women to participate. If you are a female of child-bearing age, you will 
be asked to undergo a urine pregnancy test provided by the research site prior to participating in 
any of the actual study days. Acceptable methods of birth control include oral/topical/injected 
contraceptives, intra-uterine devices, and barrier methods. If you become pregnant while taking 
part in the study, you must immediately tell your research doctor and you will be withdrawn from 
the study. We will follow the outcome of your pregnancy and we will continue to follow you 
according to the study plan. 
Exercise Risks: There is elevated risk for injury or cardiovascular events during exercise. 
However, the laboratory’s significant amount of work with exercise in healthy, diseased, and aged 
populations over the past 20 years has revealed that this is an issue of caution and when 
approached as such, is not a problem. Exercise PREHAB and REHAB will be performed under 
strict supervision in a controlled environment (please refer to following section pertaining to 
protection against risk during exercise protocols). 
 
Radiation Risks DEXA Scan: This research study involves up to 3 DEXA scans. These scans are 
not standard of care and you are receiving them only because you are enrolled in this research 
study. These procedures will expose you to radiation. The risk from this radiation exposure is 
considered to be small and comparable to other every day risks. To give you an idea of how much 
radiation you will receive, we will compare this radiation to the radiation that you receive from 
natural sources. Everyone receives a small amount of unavoidable radiation every day. Some of 
this radiation comes from space while some comes from radiation that is naturally occurring in 
water, soil, rocks and minerals found in plants and animals. The excess radiation that you will be 

exposed to in this research study is equivalent to about 2 days of natural background 
radiation.  This amount does not include any radiation exposures that you may receive from other 
types of tests 
 
RISK MANAGEMENT PROCEDURES 
By far the most important issue is prevention of the above risks by proper participant pre-screening, 
by high quality technique of catheter and by careful monitoring during exercise (see methods 
section), and by ensuring post-study patient education and follow-up of catheter site care.  For all 
protocols, ECG activity and arterial blood pressure are monitored by an observer who is not involved 
in any other aspects of the study.  Both this observer and the participant have absolute authority to 
terminate a study at any time. Participant confidentiality is ensured by the coding of all personal 
information and the maintenance of all records and samples within a locked structure accessible 
only to key personnel involved in the research study.  Furthermore, during the exercise, RPEs will 
be obtained from the participants at the end of each stage. This includes evaluation of a) overall 
feeling, b) leg discomfort, and c) dyspnea. The laboratory is equipped with a fully stocked 
resuscitation cart that includes bag/mask ventilation equipment, intubation equipment, suction 
equipment, electrical defibrillator an O2 source and resuscitation drugs including naloxone. In the 
event of symptoms or signs of an adverse cardiopulmonary event – anginal symptoms, extreme 
dyspnea, dizziness, pre-syncope or frank syncope, severe respiratory distress, decrease in SpO2 
below 90%, tachydysrythmia – exercise will be ceased immediately, with institution of evaluation 
and management according to ACLS protocol (maintenance of airway, supplemental O2, 
ventilatory assistance if necessary, electrical cardioversion or defibrillation if necessary). If the 
participant requires cardiopulmonary resuscitation or further medical therapy, they will be 
immediately transported to the Emergency Department. 
 
STATISTICAL METHODS, DATA ANALYSIS AND INTERPRETATION: 
A total of 150 participants will be recruited for the protocols outlined above. The experimental 
design and statistical analyses have been developed in collaboration with the Study Design & 
Biostatistics Center at the University of Utah. We focus on well-powered testing of the primary 
hypotheses while minimizing subject burden and study costs. Descriptive summaries will be 
provided for each outcome prior to initiating formal statistical analyses. Transformations may be 
sought for outcomes exhibiting substantial skewness to better approximate normality. If 
approximate normality cannot be achieved following transformation, rank-based non-parametric 
analyses (such as Wilcoxon rank-sum test) will be substituted for parametric analyses. 
Comparisons between conditions (ie; inactivity verse activity, placebo verse intervention, trained 
verse untrained) may be made prior to, during, and after the inactivity intervention. No baseline 
group differences are expected while significant improvements in vascular and mitochondrial 
function are expected prior to inactivity in those participants undergoing exercise training. During 
inactivity, measures of vascular and skeletal muscle function are expected to decline. Rates of 
decline are expected to be attenuated when an intervention such as exercise training or 
antioxidant supplementation (MitoQ, Protandim, PB125, Oral Antioxidant) is administered. A rich 
set of measures will be obtained from biopsied muscle, blood samples, and measures of strength, 
lean mass, and aerobic capacity before and after the intervention for each group. These 
comparisons with different outcome measures will be considered secondary or supportive 
analyses. For the formal test of the hypotheses we will use analysis of covariance (as 
implemented SAS PROC GLM) with the final vascular and mitochondrial function assessments 

as the dependent variable and baseline as independent variables as suggested by Vickers and 
Altman 69. We will also include age and gender as independent variables in the model. Similar 
models will be constructed for secondary or supportive analyses listed above.  
Clinical Records: Patient demographics (DOB, gender, height, weight), past medical history, 
medication use, and results of cardiovascular tests (echocardiogram, catheterization, stress test) 
will be collected. 
The following procedures are in place in the Vascular Laboratory to assure safety of 
research participants: 
1. Properly trained research personnel will monitor all exercises to ensure they are 
performed correctly. 
2. Individuals performing blood draws are familiar with all disease transmission 
safeguards and emergency procedures, should a problem arise. 
3. Blood pressure cuffs will only be inflated to obtain the necessary measurements, and 
will be deflated immediately afterwards. 
4. Immediate emergency – call 911, as typically employed at the Salt Lake City VA 
Medical Center that are not covered by the code team from the hospital. 
5. A crash cart and defibrillator are maintained in the laboratory. 
 
The following procedures are in place in the Vascular Laboratory to protect sensitive 
participant information: 
1. The laboratory is kept locked when there are no lab personnel present. 
2. The Logic 7 is turned off when not in use for vascular analyses. The computer is 
password protected and only the investigators related to this project know the 
password. 
3. Hard copies of participant information will be kept in locked file cabinet and locked in 
offices. Any electronic copies will be kept on a password secured computer. 
4. Participants are given copies of their measurements on request. 
5. Each participant will be identified by code numbers. These numbers will be used in a 
statistical spreadsheet while all statistical analyses are performed. The maximal length 
of storing this information is 5 years, upon which it will be destroyed.  
 
ADMINISTRATIVE RESPONSIBILITIES:  
Facilities and Equipment 
Utah Vascular Research Laboratory (UVRL): The PI, Dr. Joel Trinity, is a member of the UVRL, 
a consortium that is affiliated with the University of Utah (Department of Internal Medicine) and 
housed at the Salt Lake City Veterans Administration Medical Center Geriatrics, Research, 
Education and Clinical Center (SLC VAMC GRECC). At present, the UVRL includes 5 full-time 
faculty members, 8 postdoctoral fellows, 8 graduate students, 2 full-time laboratory specialists, 
and 2 clinical research study coordinators. The full-time laboratory specialists include a 
microbiologist and a biochemist that conduct and assist trainees in the performance of various 
assays including the biochemical analysis of free radicals and their footprints. Additionally, two 
full-time research study coordinators are responsible for scheduling and recruiting subjects and 
maintaining compliance with the University and VA Institutional Review Boards. 
 
Research performed in the UVRL revolves around the link between vascular and skeletal muscle 
function. Specifically, faculty of the UVRL, are interested in the mechanisms that appear to limit 

skeletal muscle function as the result of healthy aging and age-related disease including heart 
failure, hypertension, and chronic obstructive pulmonary disease. One of the many potential 
candidates upon which we are currently focusing considerable attention is the role of oxidative 
stress in the regulation/dysregulation of skeletal muscle metabolism and vascular control. The 
UVRL consists of 4 human-based laboratories, each approximately 800 sq. ft., and 3 wetlabs 
ranging from 250 to 800 sq. ft. All laboratories are located at the Salt Lake City VA Medical Center, 
Building 2. The proposed vascular function assessments will take place in the human based 
laboratories while chemical analysis including electron paramagnetic resonance spectroscopy, 
molecular biology, and biochemistry will be performed in 1 of the 3 wetlabs.  
 
Skeletal Muscle Exercise Research Facility (SMERF): Drs. Paul LaStayo and Micah 
Drummond (Sub-investigator’s) Co-Direct the SMERF, a clinical research training environment 
focused on the interrelationship of impaired muscle strength/power and activation; and 
mobility/gait disorders and falls. Current research performed at the SMERF is focused on aging 
muscle and how it impacts movement and human performance; specifically how aging muscles 
adapt and how these changes help or hinder human function in daily life and following injury or 
disease. The SMERF, a compilation of 5000 ft2 of clinical-lab space, is located on the 1st floor in 
the College of Health’s Department of Physical Therapy and Athletic Training. Previous and 
current clinical trials designed to evaluate muscle countermeasures and commensurate muscle, 
mobility and quality of life endpoints have been successfully performed in the SMERF across a 
variety of older individuals and clinical populations. The SMERF is located on the University of 
Utah campus yet away from busy day-to-day traffic. Therefore, it is easily accessed by car, bus 
or via the free ParaTrans system; there is ample free parking and an accessible entrance to both 
facilities for those who are mobility impaired. The SMERF houses standard muscle and mobility 
training equipment as well as measurement lab stations for assessing muscle structure/function 
and analyses of mobility/activity performance. The proposed assessments of muscle strength will 
take place at the SMERF. 
 
Center for Clinical and Translational Science (CCTS): The proposed project will utilize the 
several services provided by the University of Utah CCTS. Briefly, the CCTS will provide 24 hour 
care of subjects during the 1-10 days of bed rest. Moreover, assessment of body composition via 
Magnetic Resonance Imaging (MRI), muscle biopsies, and venous blood collections will be 
performed by CCTS nursing staff. Members of the investigative team (Trinity, Drummond, 
LaStayo, Supiano) have successfully performed and are currently performing several bed rest 
studies at the CCTS. 
 
University Center for Advanced Imaging Research (UCAIR): For studies involving NMR and 
MRI, we will collaborate with UCAIR. This center consists of a 7000 sq. ft. of office, class room 
and laboratory space located in the Center for Advanced Medical Technologies (CAMT) at 
Arapeen Drive in Research Park. Linux and SUN workstations, computer terminals, PCs and 
MacIntosh computers are available throughout the lab. The lab contains an electronics shop fully 
equipped with oscilloscopes, signal generators, and other essential test and measurement 
equipment with bench space and various equipment for electronic fabrication and testing, a small 
machine shop, and wet-lab space with fume hood in addition to a full stock of electronic parts, 
manuals, and data books. UCAIR also has a coil lab, equipped with an HP 8752A network 
analyzer, a HP 4193A vector impedance meter, and a full size test bore/RF shield to simulate the 
MRI electrical environment. The School of Medicine machine shop is located in the CAMT building 
and is fully equipped and available for major machining projects. There is approximately 800 sq. 
ft. of shielded small bore magnet space, and about 200 sq. ft. of preparation area adjacent to the 
human imaging magnets. The CAMT building also houses two Siemens Trio 3T MR scanners. 
The scanners have actively shielded water cooled Sonata gradient systems (40 mT/m amplitude, 

200 T/m/s slew rate), 32 high speed RF receiver channels and RF body coil, head coil and several 
coils of variable numbers of elements designed for specific body parts. Custom coils for unique 
geometries may be designed and fabricated in the coil development lab (above). The systems 
are also equipped with pulse sequences and appropriate software for cardiac imaging, 
spectroscopy, functional MRI, diffusion tensor MRI and other high end imaging capabilities. A 
highly flexible pulse sequence development platform (IDEA) is also included to allow research 
and development in novel techniques. We have a research agreement and research support from 
Siemens and collaborate with them. 
 
Personnel 
All investigators of this study have extensive experience with each of the techniques described in 
the proposal. 
 
Recruitment & Participant Compensation   
Participants will be recruited by staff at the Utah Vascular Research Laboratory. Recruitment 
strategies will include recruitment fliers (VAMC SLC and University of Utah), VA and University 
patient databases, newspaper advertisements, clinic visits for recruitment with the approval of the 
candidate’s MD, word of mouth. A formal analysis of electronic medical records using appropriate 
ICD-9 codes and drug indices may be used to identify eligible participants. Electronic medical 
records from both the VAMC SLC and University of Utah are currently available to the 
investigative team and will be used in this analysis. Following identification of eligible participants 
a direct mail campaign will take place in which all eligible participants will receive a letter from the 
research team stating the purpose of the study, the requirements of the study, study 
compensation, contact information, and information to opt-out from further contact and 
participation. Eligible participants that expressed interest in the study or chose not to opt-out from 
further communication will be called by a member of the research recruitment team.  
Participant compensation:  
• 
Preliminary testing and screening: Participants will receive $20/hour compensation per 
visit. 
• 
Bed rest protocol: $150 per/day of bed rest  
• 
Limb immobilization protocol: $60 per day of limb immobilization 
• 
Reduced activity Protocol: $50 per day of reduced activity 
• 
Exercise training session performed before, during or after inactivity: $20 per session. 
Communication Plans for Multi-Center Studies (i.e. multiple sites around the nation): N/A. 
 
Participating Sites outside the University of Utah (i.e. multiple sites around the city or 
state): N/A. 
 
Recordkeeping: 

Per 21 CFR 312.57, Investigator records shall be maintained for a period of 2 years following 
the date a marketing application is approved; or, if no application is filed or the application is not 
approved, until 2 years after the investigation is discontinued and the FDA is notified.   
VA Records Retention:  
The VA does not have included in their record control schedule a timeframe for the destruction 
of research records, as a result, we cannot destroy anything right now and need to ask if it is 
indicated in the application or supporting documents that records, including identifiers, will be 
destroyed within a specific timeframe that this timeframe be changed to "in accordance with the 
VA record control schedule."  
Protocol Deviation: 
A protocol deviation (or violation) is any departure from the defined procedures and 
treatment plans as outlined in the protocol version submitted and previously approved by the 
IRB. Protocol deviations have the potential to place participants at risk and can also 
undermine the scientific integrity of the study thus jeopardizing the justification for the 
research.  Protocol deviations are unplanned and unintentional events.  
 
Because some protocol deviations pose no conceivable threat to participant safety or 
scientific integrity, reporting is left to the discretion of the PI within the context of the 
guidelines below. The IRB requires the prompt reporting of protocol deviations which 
are: 
• 
Exceptions to eligibility criteria. 
• 
Intended to eliminate apparent immediate hazard to a research participant or  
• 
Harmful (caused harm to participants or others, or place them at increased 
risk of harm - including physical, psychological, economic, or social harm), or  
• 
Possible serious or continued noncompliance  
 
Protocol violations/deviations will be summarized in the FDA Annual IND Report or if safety 
issues arise they may be reported earlier. 
Prompt reporting for VA research is 5 business days upon becoming aware of a reportable 
problem, event, or deviation.  Unanticipated Problems (including Adverse Events) 
• 
The VA does not use the OHRP term of an unanticipated problem involving risks to 
participants or others.  However, the VA uses criteria for reporting problems and events that 
are nearly exact when compared to the criteria for an unanticipated problem involving risks 
to participants or others, which is used by the University of Utah IRB.  Thus, investigators 
conducting VA research should follow the standard Unanticipated Problem criteria for 
evaluating and reporting problems and events. 
• 
The investigator must notify the IRB same-day by email upon becoming aware of any 
local research-related deaths that are unanticipated.  Send notification, including the IRB 
number, to: irb@hsc.utah.edu.  The investigator must then submit a Report Form within 5 
business days.    

• 
Investigators must take care in documenting appropriate evaluation of unanticipated 
problems.  Unfounded classification of problems as unrelated or anticipated must be 
reviewed by the IRB for possible serious non-compliance. 
 
Protocol Amendments: 
Any amendments or administrative changes in the research protocol during the period, 
for which the IRB approval has already been given, will not be initiated without 
submission of an amendment for IRB review and approval.   All amendments that 
significantly affects the patient safety will be submitted to the FDA for review per 21 CFR 
312.30. These requirements for approval will in no way prevent any immediate action 
from being taken by the investigator in the interests of preserving the safety of all 
patients included in the trial.  
 
REFERENCES AND APPENDICES:  
 
1. 
Booth FW and Criswell DS. Molecular events underlying skeletal muscle atrophy 
and the development of effective countermeasures. Int J Sports Med. 1997;18 Suppl 
4:S265-9. 
2. 
Wall BT, Dirks ML, Snijders T, Senden JM, Dolmans J and van Loon LJ. 
Substantial skeletal muscle loss occurs during only 5 days of disuse. Acta Physiol (Oxf). 
2014;210:600-11. 
3. 
Restaino RM, Holwerda SW, Credeur DP, Fadel PJ and Padilla J. Impact of 
prolonged sitting on lower and upper limb micro- and macrovascular dilator function. Exp 
Physiol. 2015;100:829-38. 
4. 
English KL and Paddon-Jones D. Protecting muscle mass and function in older 
adults during bed rest. Curr Opin Clin Nutr Metab Care. 2010;13:34-9. 
5. 
Widlansky ME, Gokce N, Keaney JF, Jr. and Vita JA. The clinical implications of 
endothelial dysfunction. J Am Coll Cardiol. 2003;42:1149-60. 
6. 
Seals DR, Jablonski KL and Donato AJ. Aging and vascular endothelial function in 
humans. Clin Sci (Lond). 2011;120:357-75. 
7. 
Thompson LV. Age-related muscle dysfunction. Exp Gerontol. 2009;44:106-11. 
8. 
Proctor DN and Joyner MJ. Skeletal muscle mass and the reduction of VO2max in 
trained older subjects. Journal of applied physiology. 1997;82:1411-5. 
9. 
Powers SK, Smuder AJ and Judge AR. Oxidative stress and disuse muscle 
atrophy: cause or consequence? Curr Opin Clin Nutr Metab Care. 2012;15:240-5. 
10. 
Wall BT, Dirks ML and van Loon LJ. Skeletal muscle atrophy during short-term 
disuse: implications for age-related sarcopenia. Ageing Res Rev. 2013;12:898-906. 
11. 
Padilla J and Fadel PJ. Prolonged sitting leg vasculopathy: contributing factors and 
clinical implications. Am J Physiol Heart Circ Physiol. 2017;313:H722-H728. 
12. 
Krogh-Madsen R, Thyfault JP, Broholm C, Mortensen OH, Olsen RH, Mounier R, 
Plomgaard P, van Hall G, Booth FW and Pedersen BK. A 2-wk reduction of ambulatory 
activity attenuates peripheral insulin sensitivity. Journal of applied physiology. 
2010;108:1034-40. 

13. 
Dirks ML, Wall BT, van de Valk B, Holloway TM, Holloway GP, Chabowski A, 
Goossens GH and van Loon LJ. One Week of Bed Rest Leads to Substantial Muscle 
Atrophy and Induces Whole-Body Insulin Resistance in the Absence of Skeletal Muscle 
Lipid Accumulation. Diabetes. 2016;65:2862-75. 
14. 
Trappe TA, Burd NA, Louis ES, Lee GA and Trappe SW. Influence of concurrent 
exercise or nutrition countermeasures on thigh and calf muscle size and function during 
60 days of bed rest in women. Acta Physiol (Oxf). 2007;191:147-59. 
15. 
Donato AJ, Uberoi A, Bailey DM, Wray DW and Richardson RS. Exercise-induced 
brachial artery vasodilation: effects of antioxidants and exercise training in elderly men. 
Am J Physiol Heart Circ Physiol. 2010;298:H671-8. 
16. 
Eskurza I, Monahan KD, Robinson JA and Seals DR. Effect of acute and chronic 
ascorbic acid on flow-mediated dilatation with sedentary and physically active human 
ageing. The Journal of physiology. 2004;556:315-24. 
17. 
Taddei S, Virdis A, Ghiadoni L, Salvetti G, Bernini G, Magagna A and Salvetti A. 
Age-related reduction of NO availability and oxidative stress in humans. Hypertension. 
2001;38:274-9. 
18. 
Wray DW, Nishiyama SK, Harris RA, Zhao J, McDaniel J, Fjeldstad AS, Witman 
MA, Ives SJ, Barrett-O'Keefe Z and Richardson RS. Acute reversal of endothelial 
dysfunction in the elderly after antioxidant consumption. Hypertension. 2012;59:818-24. 
19. 
Powers SK, Hudson MB, Nelson WB, Talbert EE, Min K, Szeto HH, Kavazis AN 
and Smuder AJ. Mitochondria-targeted antioxidants protect against mechanical 
ventilation-induced diaphragm weakness. Crit Care Med. 2011;39:1749-59. 
20. 
McClung JM, Whidden MA, Kavazis AN, Falk DJ, Deruisseau KC and Powers SK. 
Redox regulation of diaphragm proteolysis during mechanical ventilation. Am J Physiol 
Regul Integr Comp Physiol. 2008;294:R1608-17. 
21. 
Min K, Smuder AJ, Kwon OS, Kavazis AN, Szeto HH and Powers SK. 
Mitochondrial-targeted antioxidants protect skeletal muscle against immobilization-
induced muscle atrophy. J Appl Physiol (1985). 2011;111:1459-66. 
22. 
Donovan EL, McCord JM, Reuland DJ, Miller BF and Hamilton KL. Phytochemical 
activation of Nrf2 protects human coronary artery endothelial cells against an oxidative 
challenge. Oxid Med Cell Longev. 2012;2012:132931. 
23. 
Seals DR, Desouza CA, Donato AJ and Tanaka H. Habitual exercise and arterial 
aging. J Appl Physiol (1985). 2008;105:1323-32. 
24. 
DeSouza CA, Shapiro LF, Clevenger CM, Dinenno FA, Monahan KD, Tanaka H 
and Seals DR. Regular aerobic exercise prevents and restores age-related declines in 
endothelium-dependent vasodilation in healthy men. Circulation. 2000;102:1351-7. 
25. 
Ji LL, Leeuwenburgh C, Leichtweis S, Gore M, Fiebig R, Hollander J and Bejma J. 
Oxidative stress and aging. Role of exercise and its influences on antioxidant systems. 
Ann N Y Acad Sci. 1998;854:102-17. 
26. 
Cornelissen VA and Fagard RH. Effects of endurance training on blood pressure, 
blood pressure-regulating mechanisms, and cardiovascular risk factors. Hypertension. 
2005;46:667-75. 
27. 
Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL, Jr., Jones 
DW, Materson BJ, Oparil S, Wright JT, Jr., Roccella EJ, Joint National Committee on 
Prevention DE, Treatment of High Blood Pressure. National Heart L, Blood I and National 
High Blood Pressure Education Program Coordinating C. Seventh report of the Joint 

National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood 
Pressure. Hypertension. 2003;42:1206-52. 
28. 
Pescatello LS, Franklin BA, Fagard R, Farquhar WB, Kelley GA, Ray CA and 
American College of Sports M. American College of Sports Medicine position stand. 
Exercise and hypertension. Med Sci Sports Exerc. 2004;36:533-53. 
29. 
Ploutz-Snyder LL, Downs M, Ryder J, Hackney K, Scott J, Buxton R, Goetchius E 
and Crowell B. Integrated resistance and aerobic exercise protects fitness during bed 
rest. Med Sci Sports Exerc. 2014;46:358-68. 
30. 
Fisher SR, Goodwin JS, Protas EJ, Kuo YF, Graham JE, Ottenbacher KJ and Ostir 
GV. Ambulatory activity of older adults hospitalized with acute medical illness. J Am 
Geriatr Soc. 2011;59:91-5. 
31. 
Fisher SR, Kuo YF, Graham JE, Ottenbacher KJ and Ostir GV. Early ambulation 
and length of stay in older adults hospitalized for acute illness. Archives of internal 
medicine. 2010;170:1942-3. 
32. 
Vigelsø A, Gram M, Wiuff C, Andersen JL, Helge JW and Dela F. Six Weeks' 
Aerobic Retraining After Two Weeks' Immobilization Restores Leg Lean Mass and 
Aerobic Capacity But Does Not Fully Rehabilitate Leg Strength in Young and Older Men. 
Journal of rehabilitation medicine. 2015;47:552-560. 
33. 
Yasuda N, Glover EI, Phillips SM, Isfort RJ and Tarnopolsky MA. Sex-based 
differences in skeletal muscle function and morphology with short-term limb 
immobilization. Journal of Applied Physiology. 2005;99:1085-1092. 
34. 
Berg H and Tesch P. Changes in muscle function in response to 10 days of lower 
limb unloading in humans. Acta Physiologica. 1996;157:63-70. 
35. 
Jones SW, Hill RJ, Krasney PA, O’Connner B, Peirce N and Greenhaff PL. Disuse 
atrophy and exercise rehabilitation in humans profoundly affects the expression of genes 
associated with the regulation of skeletal muscle mass. The FASEB Journal. 
2004;18:1025-1027. 
36. 
Breen L, Stokes KA, Churchward-Venne TA, Moore DR, Baker SK, Smith K, 
Atherton PJ and Phillips SM. Two weeks of reduced activity decreases leg lean mass and 
induces "anabolic resistance" of myofibrillar protein synthesis in healthy elderly. J Clin 
Endocrinol Metab. 2013;98:2604-12. 
37. 
Morishima T, Restaino RM, Walsh LK, Kanaley JA, Fadel PJ and Padilla J. 
Prolonged sitting-induced leg endothelial dysfunction is prevented by fidgeting. Am J 
Physiol Heart Circ Physiol. 2016;311:H177-82. 
38. 
Restaino RM, Walsh LK, Morishima T, Vranish JR, Martinez-Lemus LA, Fadel PJ 
and Padilla J. Endothelial dysfunction following prolonged sitting is mediated by a 
reduction in shear stress. Am J Physiol Heart Circ Physiol. 2016;310:H648-53. 
39. 
Benzie IF and Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure 
of "antioxidant power": the FRAP assay. Anal Biochem. 1996;239:70-6. 
40. 
Bailey DM, Young IS, McEneny J, Lawrenson L, Kim J, Barden J and Richardson 
RS. Regulation of free radical outflow from an isolated muscle bed in exercising humans. 
Am J Physiol Heart Circ Physiol. 2004:00148.2004. 
41. 
Richardson RS, Donato AJ, Uberoi A, Wray DW, Lawrenson L, Nishiyama S and 
Bailey DM. Exercise-induced brachial artery vasodilation: role of free radicals. Am J 
Physiol Heart Circ Physiol. 2007;292:H1516-22. 

42. 
Nishiyama SK, Wray DW and Richardson RS. Sex and limb-specific ischemic 
reperfusion and vascular reactivity. Am J Physiol Heart Circ Physiol. 2008;295:H1100-
H1108. 
43. 
Wray DW, Uberoi A, Lawrenson L, Bailey DM and Richardson RS. Oral 
antioxidants and cardiovascular health in the exercise-trained and untrained elderly: a 
radically different outcome. Clin Sci (Lond). 2009;116:433-41. 
44. 
Wray DW, Nishiyama SK, Monnet A, Wary C, Duteil SS, Carlier PG and 
Richardson RS. Antioxidants and aging: NMR-based evidence of improved skeletal 
muscle perfusion and energetics. Am J Physiol Heart Circ Physiol. 2009;297:H1870-5. 
45. 
Harris RA, Nishiyama SK, Wray DW, Tedjasaputra V, Bailey DM and Richardson 
RS. The effect of oral antioxidants on brachial artery flow-mediated dilation following 5 
and 10 min of ischemia. Eur J Appl Physiol. 2009;107:445-53. 
46. 
Clark RV, Walker AC, O'Connor-Semmes RL, Leonard MS, Miller RR, Stimpson 
SA, Turner SM, Ravussin E, Cefalu WT, Hellerstein MK and Evans WJ. Total body 
skeletal muscle mass: estimation by creatine (methyl-d3) dilution in humans. J Appl 
Physiol (1985). 2014;116:1605-13. 
47. 
Hogikyan RV and Supiano MA. Arterial alpha-adrenergic responsiveness is 
decreased and SNS activity is increased in older humans. Am J Physiol. 1994;266:E717-
24. 
48. 
Wray DW, Nishiyama SK, Harris RA and Richardson RS. Angiotensin II in the 
elderly: impact of angiotensin II type 1 receptor sensitivity on peripheral hemodynamics. 
Hypertension. 2008;51:1611-6. 
49. 
Wray DW, Nishiyama SK, Donato AJ, Sander M, Wagner PD and Richardson RS. 
Endothelin-1-mediated vasoconstriction at rest and during dynamic exercise in healthy 
humans. Am J Physiol Heart Circ Physiol. 2007;293:H2550-6. 
50. 
Wray DW, Fadel PJ, Smith ML, Raven P and Sander M. Inhibition of alpha-
adrenergic vasoconstriction in exercising human thigh muscles. J Physiol. 2004;555:545-
63. 
51. 
Brothers RM, Haslund ML, Wray DW, Raven PB and Sander M. Exercise-induced 
inhibition of angiotensin II vasoconstriction in human thigh muscle. J Physiol. 
2006;577:727-37. 
52. 
Hogikyan RV and Supiano MA. Homologous upregulation of human arterial alpha-
adrenergic responses by guanadrel. J Clin Invest. 1993;91:1429-35. 
53. 
Barden J, Lawrenson L, Poole JG, Kim J, Wray DW, Bailey DM and Richardson 
RS. Limitations to vasodilatory capacity and VO2 max in trained human skeletal muscle. 
Am J Physiol Heart Circ Physiol. 2007;292:H2491-7. 
54. 
Richardson RS, Grassi B, Gavin TP, Haseler LJ, Tagore K, Roca J and Wagner 
PD. Evidence of O2 supply-dependent VO2 max in the exercise-trained human 
quadriceps. J Appl Physiol. 1999;86:1048-53. 
55. 
Lawrenson L, Hoff J and Richardson RS. Aging attenuates vascular and metabolic 
plasticity but does not limit improvement in muscle VO(2) max. Am J Physiol Heart Circ 
Physiol. 2004;286:H1565-72. 
56. 
Richardson RS, Leek BT, Gavin TP, Haseler LJ, Mudaliar SR, Henry R, Mathieu-
Costello O and Wagner PD. Reduced mechanical efficiency in chronic obstructive 
pulmonary disease but normal peak VO2 with small muscle mass exercise. Am J Respir 
Crit Care Med. 2004;169:89-96. 

57. 
de Wilde RB, Geerts BF, Cui J, van den Berg PC and Jansen JR. Performance of 
three minimally invasive cardiac output monitoring systems. Anaesthesia. 2009;64:762-
9. 
58. 
Sugawara J, Tanabe T, Miyachi M, Yamamoto K, Takahashi K, Iemitsu M, Otsuki 
T, Homma S, Maeda S, Ajisaka R and Matsuda M. Non-invasive assessment of cardiac 
output during exercise in healthy young humans: comparison between Modelflow method 
and Doppler echocardiography method. Acta Physiol Scand. 2003;179:361-6. 
59. 
Bailey DM, Ainslie PN, Jackson SK, Richardson RS and Ghatei M. Evidence 
against redox regulation of energy homoeostasis in humans at high altitude. Clin Sci 
(Lond). 2004;107:589-600. 
60. 
Bailey DM, Young IS, McEneny J, Lawrenson L, Kim J, Barden J and Richardson 
RS. Regulation of free radical outflow from an isolated muscle bed in exercising humans. 
Am J Physiol Heart Circ Physiol. 2004;287:H1689-1699. 
61. 
Donato AJ, Uberoi A, Bailey DM, Walter Wray D and Richardson RS. Exercise-
induced brachial artery vasodilation: effects of antioxidants and exercise training in elderly 
men. Am J Physiol Heart Circ Physiol. 2010;298:H671-678. 
62. 
Donato AJ, Eskurza I, Silver AE, Levy AS, Pierce GL, Gates PE and Seals DR. 
Direct Evidence of Endothelial Oxidative Stress With Aging in Humans: Relation to 
Impaired Endothelium-Dependent Dilation and Upregulation of Nuclear Factor-{kappa}B. 
Circ Res. 2007;100:1659-1666. 
63. 
Smith RA and Murphy MP. Animal and human studies with the mitochondria-
targeted antioxidant MitoQ. Ann N Y Acad Sci. 2010;1201:96-103. 
64. 
Snow BJ, Rolfe FL, Lockhart MM, Frampton CM, O'Sullivan JD, Fung V, Smith RA, 
Murphy MP, Taylor KM and Protect Study G. A double-blind, placebo-controlled study to 
assess the mitochondria-targeted antioxidant MitoQ as a disease-modifying therapy in 
Parkinson's disease. Mov Disord. 2010;25:1670-4. 
65. 
Gane EJ, Weilert F, Orr DW, Keogh GF, Gibson M, Lockhart MM, Frampton CM, 
Taylor KM, Smith RA and Murphy MP. The mitochondria-targeted anti-oxidant 
mitoquinone decreases liver damage in a phase II study of hepatitis C patients. Liver Int. 
2010;30:1019-26. 
66. 
Hennessey JV, Chromiak JA, DellaVentura S, Guertin J and MacLean DB. 
Increase in percutaeous muscle biopsy yield with a suction-enhancement technique. J 
Appl Physiol. 1997;82:1739-1742. 
67. 
Reuland DJ, Khademi S, Castle CJ, Irwin DC, McCord JM, Miller BF and Hamilton 
KL. Upregulation of phase II enzymes through phytochemical activation of Nrf2 protects 
cardiomyocytes against oxidant stress. Free Radic Biol Med. 2013;56:102-11. 
68. 
Konopka AR, Laurin JL, Musci RV, Wolff CA, Reid JJ, Biela LM, Zhang Q, Peelor 
FF, 3rd, Melby CL, Hamilton KL and Miller BF. Influence of Nrf2 activators on subcellular 
skeletal muscle protein and DNA synthesis rates after 6 weeks of milk protein feeding in 
older adults. Geroscience. 2017;39:175-186. 
69. 
Vickers AJ and Altman DG. Analysing controlled trials with baseline and follow up 
measurements. Bmj. 2001;323:1123-1124.