Optimizing Protein Intake in Older Americans With Mobility Limitations
Phase: PHASE3
Conditions: Mobility Limitation
Sponsor: Brigham and Women's Hospital

 
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TITLE:  OPTIMIZING PROTEIN INTAKE IN OLDER AMERICANS WITH 
MOBILITY LIMITATIONS 
THE STUDY PROTOCOL 
PRINCIPAL INVESTIGATOR:  
 
SHALENDER BHASIN, MD 
JOINT PI:  
 
 
 
 
CAROLINE APOVIAN, MD 
CO-INVESTIGATORS: 
LYNN MOORE, TOM TRAVISON, 
THOMAS W. STORER, 
SHEHZAD BASARIA, ALAN 
JETTE 
FUNDING:  
 
 
 
 
NATIONAL INSTITUTE ON   
 
 
 
 
 
 
 
AGING 
NIA GRANT NUMBER: 
 
 
1R01AG037547-01 
APPLICANT ORGANIZATION: 
BRIGHAM 
AND 
WOMEN’S 
HOSPITAL 
 
VERSION: 5.3 
 
DATE:  08/23/2016 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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TABLE OF CONTENTS 
 
1. STUDY OBJECTIVES 
7 
2. INTRODUCTION 
2.1 
Background 
 
8-13 
3. STUDY DESIGN 
13 
4. OUTCOME MEASURES 
4.1 
Primary Outcome Measure 
4.2 
Secondary Outcome Measures 
4.3 
Safety measures 
5. SUBJECT SELECTION AND WITHDRAWAL 
5.1.         Number of Subjects 
5.2 
Inclusion Criteria 
5.2 
Exclusion Criteria  
5.3 
Subject Recruitment and Screening 
5.4 
Early Withdrawal of Subjects 
5.5 
Restrictions during the course of the study 
13-14 
13 
14 
14 
14 
14 
15 
15-16 
16 
16 
16 
6. STUDY DRUG  
5.1 
Description 
5.2. 
Treatment Regimen 
5.3 
Drug Storage 
5.4 
Subject Randomization and Stratification 
5.5.  
Blinding 
17 
17 
17 
17 
17 
17 
7. STUDY PROCEDURES AND VISITS 
7.1 
Telephone Screening  
7.2 
Visit 1 (V1) 
7.3 
Visit 2 (V2) 
7.4 
The Run-in-Period 
7.5 
Baseline Visit 
7.6 
Randomization 
7.7 
Intervention Period 
7.8 
Subject Compensation 
18 
18 
18 
18 
19 
19 
19 
20 
24 
8. 
DIETARY PRESCRIPTION, AND ENHANCING AND VERIFYING DIETARY 
COMPLIANCE 
8.1 
Standardizing energy and protein content through packaged meals and 
24-26 
 
24 

 
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supplement 
8.2 
Preparation of the Dietary Supplements 
8.3. 
Compliance with Dietary Prescription 
8.4. 
Exercise and Activity                                                                                            
 
25 
26 
9. STATISTICAL PLAN 
9.1 
Analytical Methods and Sample Size Estimations 
9.1.1 
Statistical Analyses 
9.1.7 
Sample Size Estimate 
9.1.8 
Effect of Drop-out Rate 
26-31 
26-31 
26-29 
29 
31 
10. ADVERSE EVENTS  
10.1 
Interim monitoring 
10.2 
Safety and Adverse Events 
10.2.1 
Definitions 
10.2.2 
Adverse Event 
10.2.3 
Serious Adverse Event  
10.2.4 
Unanticipated Problem 
10.2.5 
Adverse Event Reporting Period  
10.2.6 
Preexisting Condition 
10.2.7 
General Physical Examination Findings  
10.2.8 
Post-study Adverse Event 
10.2.9 
Abnormal Laboratory Values 
10.2.10 Hospitalization, Prolonged Hospitalization or Surgery 
10.3 
Recording of Adverse Events 
10.4 
Reporting of Serious Adverse Events 
10.4.1 
Study Sponsor Notification by Investigator 
10.4.2 
Termination Criteria 
10.4.3 
Unblinding 
31-41 
31 
31 
32 
32 
32 
32 
33 
33 
 
33 
33 
 
33 
33 
34 
11. MONITORING SUBJECT SAFETY 
11.1 
Potential Risks to Subjects 
11.2 
Procedures for Protecting Against Potential Risks 
35-41 
35-41 
41 
11.3.       Data and Safety Monitoring Board 
12. DATA MANAGEMENT 
12.1 
Design Randomization through a Web Application 
12.2 
Design database for subject enrollment and tracking 
12.3 
Design data collection forms 
41 
42-43 
42 
42 
 

 
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12.4 
Data Capture and Quality Control 
12.5 
Dietary data 
12.6 
Create analytic datasets 
12.7 
Data Security 
12.8 
Maintaining Blinding 
12.9 
Maintaining Anonymity of Submitted Medical Records 
12.10 
Confidentiality 
42 
42 
42 
43 
43 
43 
43 
13. REFERENCES 
44 

 
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Study Summary 
Title 
Optimizing Protein Intake in Older Americans (OPTIMen) 
Protocol Number 
1R01AG037547-01 
Study Design 
Randomized, placebo-controlled, parallel group, double-blind 
Study Duration 
Five years 
Study Center 
Single site: Brigham and Women’s Hospital     
Objectives 
To determine whether administration of 1.3 g • kg-1 • day-1 of 
protein results in greater improvements than the RDA (0.8 g • kg-1 
• day-1) in lean body mass, maximal voluntary muscle strength 
and power, and self-reported and performance-based measures 
of physical function in older men whose daily protein intake is 
less than the RDA and in whom energy intake has been 
standardized at 30 kcal• kg-1 • day-1 
Number of 
Subjects 
152 
Main Inclusion 
Criteria 
Community dwelling, older men, 65 years of age or older, who 
have SPPB scores between 3 and 10, and protein intake 
<0.83/g/day  
Study Product, 
Dose, Route, 
Regimen 
Testosterone enanthate in sesame oil 100 mg intramuscularly 
weekly 
Duration of 
administration 
6 months 
Statistical 
Methodology 
Our primary outcome is change in lean body mass from baseline 
to 6 months assessed by using dual energy X-ray absorptiometry 
(DXA). Our primary analytical strategy is to use mixed-effects 
regression analysis to assess 3 and 6 month outcomes 
simultaneously with baseline total body lean mass considered as 
a covariate, as is appropriate for between-group comparisons of 
randomized subcohorts, and 6-month differences between arms 
will be estimated via a treatment contrast and corresponding 95% 
confidence interval. 

 
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1. 
Study Objectives 
The recommended dietary allowance (RDA) for protein has been set at 0.8 
grams/kg/day; this allowance is intended to meet the needs of essentially the entire 
adult population – men and women, young and old (1). However, the protein 
requirements in older individuals remain poorly validated and have engendered 
vigorous debate (2-6). Many experts advocate dietary protein intakes substantially 
above the current RDA to help maintain muscle anabolism in older individuals; these 
scientists cite epidemiological studies and intervention trials which suggest that protein 
intakes above the current RDA (0.8 g • kg-1 • day-1) for older adults have beneficial 
anabolic effects on the skeletal muscle (2, 3, 7-17). Other experts have expressed 
strong dissenting views and support the current protein RDA (4, 5). The efficacy of 
protein intakes in excess of the RDA in improving skeletal muscle mass, muscle 
performance and physical function has not been demonstrated in older individuals and 
is an issue of enormous public health and policy implications. 
An exhaustive survey of nutrient intakes in approximately 50,000 Americans residing in 
48 states by the US Department of Agriculture revealed that about a third of older 
Americans do not ingest the RDA for protein (18); more recent surveys have confirmed 
these findings (19). Low protein intake has been implicated as a remediable contributor 
to the multifactorial pathophysiology of sarcopenia, the aging-associated loss of muscle 
mass and function, which has been recognized by the Centers for Disease Control and 
National Institute on Aging as one of the major health risks facing older Americans (2, 7, 
9, 11, 13-15). However, we do not know whether increasing protein intake in older 
Americans, whose protein intake is below the RDA, increases skeletal muscle mass, 
muscle performance and physical function. Therefore, the primary objective of this 
investigation is to determine whether increasing protein intake in older Americans with 
daily protein intake less than the RDA to 1.3 g • kg-1 • day-1 will result in greater gains in 
lean body mass, muscle performance and physical function than protein intake that 
equals the RDA (0.8 g • kg-1 • day-1).  
Nitrogen balance studies in athletes have suggested that protein intake substantially 
greater than the RDA may be required to maintain nitrogen balance in individuals 
undergoing resistance exercise training and that even higher protein intake may be 
needed to achieve positive nitrogen balance and optimize adaptations to exercise 
training in these athletes.  Remarkably, however, the studies in older individuals have 
failed to demonstrate unequivocal improvements in lean body mass or physical function 
in older individuals fed higher amounts of protein in excess of the RDA. The protein 
requirements for achieving optimal anabolic response to administration of promyogenic 
function promoting therapies are unknown. Therefore, our second objective is to 
determine whether a higher level of protein intake - in excess of the RDA - is needed in 
older individuals to optimize anabolic response to a promyogenic function promoting 
anabolic therapy. 
Specific Aim 1 
To determine whether administration of 1.3 g • kg-1 • day-1 of protein results in 
greater improvements than the RDA (0.8 g • kg-1 • day-1) in lean body mass, maximal 

 
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voluntary muscle strength and power, and self-reported and performance-based 
measures of physical function in older men whose daily protein intake is less than 
the RDA and in whom energy intake has been standardized at 30 kcal• kg-1 • day-1 
 
Hypothesis 1 
 
In older individuals whose daily protein intake is less than the RDA, increasing 
dietary protein intake to 1.3 g • kg-1 • day-1 without changing the total daily energy 
intake, will not result in greater improvements in lean body mass, maximal voluntary 
strength, and self-reported as well as performance-based measures of physical 
function than ingesting the RDA.  
Specific Aim 2 
 
To determine whether the gains in lean body mass, maximal voluntary strength, and 
self-reported and performance-based measures of physical function during 
testosterone administration are greater with 1.3 g protein than the RDA in older men 
whose baseline daily protein intake is less than or equal to the RDA and in whom 
energy intake has been standardized  
 
Hypothesis 2 
 
The RDA of 0.8 g • kg-1 • day-1 protein intake is insufficient to attain optimal anabolic 
response to administration of a promyogenic function promoting therapy, such as 
testosterone. In individuals whose daily protein intake is less than the RDA, 
increasing dietary protein intake to 1.3 g • kg-1 • day-1  without changing the total 
daily energy intake, will augment anabolic response to testosterone therapy and will 
be associated with greater gains in lean body mass and maximal voluntary strength 
than the RDA. 
2. 
Introduction 
This document is a protocol for a human research study to be conducted according to 
US and international standards of Good Clinical Practice (FDA Title 21 part 312 and 
International Conference on Harmonization guidelines), applicable government 
regulations and institutional research policies and procedures.  
2.1. 
Background 
Protein requirements for older individuals have been the subject of contentious debate 
for some time. Currently, the Estimated Average Requirement (EAR) for protein (0.66 g 
protein • kg body weight-1• day-1) and the Recommended Dietary Allowance (RDA) for 
protein are the same for adult men and women of all ages (1, 20).  While the RDAs 
have assumed the force of biblical diction and guided national policy and clinical 
practice, the data on which the RDA for protein is founded have been justifiably 
criticized (2, 3, 10, 17, 21). The RDA estimate has been derived largely from short term 
studies of two to three week duration, using nitrogen balance as the outcome (1, 20).  
Numerous conceptual and methodological issues related to nitrogen balance studies (1, 
3), and additional issues related to subject selection have clouded the inferences drawn 
from these studies (3, 10). Most of the original nitrogen balance studies that formed the 

 
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basis of these recommendations were conducted after only 2-3 weeks of adaptation (4, 
20); urea turnover is slow and these short durations were likely insufficient to achieve a 
metabolic steady state (10).  The nitrogen balance studies generally used low levels of 
protein intake; adaptation to this low level of protein intake would be expected to reduce 
urinary nitrogen excretion.  Thus, the calculation of protein level required to achieve 
zero nitrogen balance would likely be underestimated.  These studies included only a 
small number of older men and women.  None of the original studies included 
measurements of lean body mass, muscle performance or physical function (1). The 
nitrogen balance studies have been criticized on multiple other grounds: inclusion of 
very few older individuals, inclusion of subjects with undocumented medical conditions, 
problems with complete urine and fecal collections, not accounting for other sources of 
nitrogen loss, and inclusion of subjects with inadequate or excessive energy intakes 
(10). Not surprisingly, the recommendations for older individuals have invited a great 
deal of disagreement; some experts have advocated higher levels of protein intake for 
older individuals (2, 6, 10, 21-27), while others have equally fervently supported the 
adequacy of the current RDA (4, 5, 20-21, 28-29).  Thus, the issue being addressed in 
this investigation has enormous public health and policy implications: if the protein 
requirements to maintain optimum muscle anabolism are higher for older individuals 
than young persons - as has been advocated by some - then a reconsideration of public 
policy and clinical practice is warranted. As a vast majority of older Americans ingest 
substantially lower amounts of protein than those being recommended by some (30-32), 
implementation of a change in protein RDA for older Americans will not be easy.  
The proposed investigation also has implications for the application of pharmacological 
function promoting anabolic therapies (FPATs), such as testosterone, selective 
androgen receptor modulators (SARMs), recombinant human growth hormone, growth 
hormone secretagogs, and myostatin antagonists.  We do not know whether protein 
intakes higher than the RDA are needed to realize optimal anabolic response to FPATs 
in older individuals. Thus, the proposed study will help determine whether protein intake 
higher than the RDA augments the anabolic response of older individuals to a 
prototypical pharmacologic anabolic intervention, namely testosterone, whose effects on 
skeletal muscle mass and strength have been well documented.  
2.1.2.  Functional Limitations and Sarcopenia in Older Americans as Major 
Societal Problems 
As men and women get older, skeletal muscle mass declines because of a decrease in 
the number and cross sectional area of muscle fibers (33-37) and a preferential loss of 
fast twitch type II fibers (38-39). The loss of muscle mass that occurs with aging is 
associated with a reduction in muscle strength and power, and an increase in muscle 
fatigability (36, 40-45). Loss of muscle mass and strength leads to impairment of 
physical function, as indicated by the impaired ability to rise from a chair, climb stairs, 
generate gait speed, and maintain balance (46-47). Limitations in physical function are 
associated with adverse health outcomes: increased risk of disability, mortality, 
hospitalization, and poor quality of life (48-59).  
The 2003 National Health Interview Survey and 2004 US Census Bureau estimate that 
12–14% of non-institutionalized population—approx. 35–38 million people—have a 

 
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disability due to a chronic health condition (60). For people over age 65, 35 to 40% 
experience activity limitations or disability. Because U.S. population is aging, the 
percentage of population aged 65 years or over will increase from 12% in 2000 to 20% 
in 2030—to over 69 million (61-63). The number of people aged 85 or over is expected 
to grow from 3 million (2.1%) to 6.2 million (3.4%). The majority of individuals who reach 
this age will experience some limitation in function. If the rate of limitations for those > 
65 were to stay at 40% through 2030, the number of older people with functional 
limitations or disability would increase to 28 million (63). The costs of support services 
and lost productivity associated with disabling conditions have consequences for the 
entire population (58, 64). The Social Security Disability Program and the Supplemental 
Security Program pay $62.5 billion yearly to 7.5 million disabled persons (63). Thus, 
there is a pressing need for function promoting anabolic therapies – nutritional, physical, 
as well as pharmacological.   
2.1.3. Multifactorial Pathophysiology of Aging-Associated Sarcopenia 
Functional limitations associated with aging are multifactorial in their pathophysiology; 
impaired protein and energy intake, androgen deficiency, abnormalities in the growth 
hormone-insulin like growth factor-1 axis, anabolic resistance, inflammation, motor 
neuron remodeling, perturbations of muscle protein dynamics, and decreased physical 
activity are all likely contributors (12, 17, 65, 66-73). Of these factors, inadequate dietary 
protein intake has been well documented to accelerate this process (12-14, 17, 74), is 
potentially reversible, and is the focus of this proposal.   
2.1.4. High prevalence of suboptimal protein intake in older Americans 
A USDA survey in the late 1970s revealed that a third of the US population of 48 states 
consumed less protein than the RDA (30).  More recent data from the 1996 Continuing 
Survey of Food Intake by Individuals largely confirmed the findings of the 1978 survey 
that ~40% of men and women, 70 years of age or older, consume less than the RDA for 
protein (0.8 g · kg–1 · d–1), and ~16% consumed less than 75% of the RDA (31). In 
addition, older adults consume inadequate amounts of good quality animal protein.  
2.1.5. Epidemiological studies of the health consequences of low protein diet  
A few cross-sectional studies that have examined the association of dietary protein 
intake and body composition in older adults have not found an association between 
protein intake with lean body mass (34, 75). In one of the most comprehensive analyses 
of the longitudinal data from the Health ABC Study, Houston et al (14) found that dietary 
protein intake was associated with changes in lean body mass during a 3-year follow-up 
period in older, community-dwelling adults. Participants in the highest quintile of energy-
adjusted protein intake lost ~40% less lean mass and appendicular skeletal muscle 
mass over the 3-year follow-up than those in the lowest quintile (14). Among individuals, 
who lost weight over the 3-year period, lower protein intake was associated with greater 
loss of lean body mass (14). A 10-year longitudinal study in New Mexico reported that 
women with protein intakes of 1.76 g protein • kg-1 • day-1 had fewer health problems 
than those consuming the RDA (76).  
2.1.6. Intervention trials of varying levels of protein intake 

 
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Several small, short term studies of 9-14 week duration have examined the effects of 
alterations in protein intake on body composition and nitrogen balance (25, 77-78). 
Castaneda et al (79) found that older women fed a weight-maintenance diet with low 
protein content remained in a negative nitrogen balance throughout the 9-week study, 
and experienced significant losses in lean body mass, immune response, and muscle 
function, and muscle fiber atrophy (78). In another diet study (11), ambulatory men and 
women, aged 55 to 77 years, who were provided eucaloric diets that contained 0.8 g 
protein·kg-1·day-1, experienced significant loss of mid-thigh muscle mass and urinary 
nitrogen excretion.  
Most studies that have compared the effects of high protein intake with the protein RDA 
have failed to find significant gains in lean body mass or muscle strength with the higher 
protein intake in spite of positive nitrogen balance with the higher protein intake (23, 80-
82). A meta-analysis of trials that investigated the effects of increased protein and 
energy intake on health outcomes found a small weight gain but no improvements in 
function or mortality (83) in subjects receiving the supplement; this meta-analysis has 
been criticized for including heterogeneous trials in widely divergent populations, 
including hospitalized patients. A long term randomized trial of high protein-energy 
supplementation in frail elderly also found no difference in lean body mass between the 
placebo and protein-supplemented groups (84).  Amino acid supplementation in some 
small studies has been shown to modestly increase muscle protein synthesis and lean 
body mass (85-87).  Thus, overall, the evidence supporting the assertion that increasing 
protein intake above the RDA increases lean body mass, strength or physical function is 
weak.     
The reasons for the striking failure of high protein intake alone to increase lean body 
mass in older individuals are not entirely clear, but several explanations are plausible. 
Most studies of high protein intake were of short duration and longer duration may be 
needed to accrue new muscle mass.  Fiatarone et al (81) found that older subjects who 
were fed a nutritional supplement reduced their intake of other foods; therefore, the net 
protein-energy intake remained unchanged. Volpi (88), Rennie (80, 89), and others 
(106, 107) have invoked an intrinsic anabolic resistance in older individuals (65, 80, 88) 
due to age-related deficits in protein kinase B/ mTOR/ elF4BP1 signaling pathways (90), 
rendering them unable to assimilate the increased protein intake into skeletal muscle 
mass accretion.  This anabolic resistance may be overcome by resistance exercise 
training or by other anabolic stimuli (80, 91). Indeed, protein intakes in excess of RDA, 
when administered in conjunction with an anabolic stimulus, such as resistance 
exercise training or testosterone, have been associated with significant gains in lean 
body mass and muscle strength (81, 92).  Trials conducted in athletes or young 
volunteers (8, 94-96) suggest that higher protein intakes enhance skeletal muscle mass 
and strength gains during resistance exercise training leading the International Society 
of Sports Nutrition to recommend in a position statement that “protein intakes of 1.4 – 
2.0 g • kg-1 • day-1 for physically active individuals are not only safe, but may improve 
the training adaptations to exercise training (8).” Taken together, these data led us to 
hypothesize that increasing dietary protein alone is insufficient to induce lean mass 
accretion, but that higher protein intake, given in the setting of an anabolic therapy such 

 
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as testosterone or resistance exercise training, would be associated with significant 
gains in lean body mass, muscle strength, and physical function.  
2.1.7. Potential Adverse Effects of High Protein Diet 
There are no long term, adequately powered studies to determine the adverse health 
consequences of eucaloric, high protein diets in older individuals. Potential risks include 
the risk of renal dysfunction, hypercalciurea, and osteoporosis. Additionally, people 
often perceive high protein diets as being “unhealthy” – containing “too much fat or 
animal products”.  For every 25-g increase in dietary protein, urinary calcium increases 
by 0.8 mmol (97). There is concern that increased calcium exertion during ingestion of 
high protein diet may reflect bone demineralization (98-99) and increase the risk of 
kidney stones (100). Increased amounts of dietary protein also might be expected to 
increase endogenous acid production (101) and bone may be called upon to buffer the 
additional acid load, resulting in mobilization of bone calcium, and a decline in bone 
mineral density (BMD). A few cross-sectional studies have reported a positive 
association between dietary protein intake and hip fracture incidence (102-103).  
However, bone balance studies have not found an increase in bone calcium resorption. 
Instead, Kerstetter et al (97) found that additional dietary protein promotes intestinal 
calcium absorption and reduces the fraction of urinary calcium of bone origin. Also at 
odds with the bone hypothesis are a large number of epidemiological studies showing 
that a high-protein diet is associated with high, not low BMD (98, 104-105). The net 
effect of dietary protein on bone mineral status depends on dietary availability of calcium 
(106); a diet rich in protein, dairy, and calcium attenuates urinary calcium and bone loss 
relative to a high carbohydrate diet (107).  
Brenner had hypothesized that high protein intakes by diabetic individuals create 
hyperfiltration and glomerular hypertension resulting in renal damage (108); however, 
this hypothesis has not found strong support (109). There is no evidence that high 
protein intakes up to 2 g ·kg–1 ·d–1 increase the risk of renal impairment in individuals 
with normal renal function (110-114).  However, in individuals with pre-existing 
impairment of renal function, high protein intake greater than 45% of energy intake may 
accelerate the decline in renal function (110, 114). The amount of protein being 
investigated in this study (0.8 and 1.3 g • kg-1 • day-1) is lower than the levels that have 
associated with renal impairment. Also, we will exclude individuals with estimated GFR 
<60 ml/min.  We will monitor serum creatinine and 24-hour calcium excretion during the 
course of the study.   
2.1.8. Enhancing Compliance with Dietary Macronutrient Interventions 
Several approaches – packaged meals, portion control, frequent contact and 
reinforcements, and frequent checks of dietary compliance in real time by using food 
compliance checklists - have proven effective in enhancing compliance with dietary 
prescriptions in nutrition intervention trials. Packaged meals and portion control are 
widely acknowledged as important contributors to improved compliance (115-116). 
Numerous trials have established the value of packaged meals, portion control, frequent 
contact and reinforcements, and frequent checks of dietary compliance in real time by 
using compliance checklists in enhancing compliance with dietary and lifestyle 

 
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interventions. Accordingly, we have incorporated all of these strategies in the design of 
the proposed trial.  
2.1.9. Effects of Testosterone on Muscle Mass, Muscle Performance, and 
Physical Function 
Several lines of evidence suggest that the decrease in testosterone with increasing age 
could be a cause of the decrease in muscle mass and strength and the decrease in 
physical function with increasing age. The men who are hypogonadal due to pituitary or 
testicular disease or due to treatment with GnRH analogs have decreased muscle mass 
(17-119).  In population studies, lower testosterone concentrations are associated with 
decreased muscle mass and strength (34-36, 120), and physical function, and 
increased risk of falls (121). In the Longitudinal Aging Study of Amsterdam and MrOS, 
serum testosterone concentrations were positively correlated with muscle strength and 
physical performance (120-121). In the Massachusetts Male Aging Study, performance 
on a physical function test was positively correlated with the serum testosterone 
concentration up to the threshold value of 461 ng/dL (122). Self-reported physical 
function, as assessed by the physical function domain of SF-36, has been associated 
with total and bioavailable testosterone levels (123). In the MMAS, both total and free T 
levels were lower in men considered frail by either slow walking or grip strength (124).  
Testosterone treatment of young hypogonadal men who have testosterone deficiency 
significantly increases lean body mass (125-129) and muscle strength (125, 129). 
Testosterone trials in men who have low testosterone levels in association with HIV 
wasting (129-132) and chronic obstructive pulmonary disease (133) also demonstrate 
increases in lean body mass and muscle strength.  
Meta-analyses of randomized controlled trials (134-135) in which older men with low or 
low-normal testosterone concentrations were treated with testosterone have 
demonstrated an increase in lean body mass (136-146), muscle strength, and self-
reported physical function (129, 133, 147). The effects on performance-based measures 
of physical function have been inconsistent across trials (137, 143, 145-146). Many 
reasons have been postulated for the inconsistency in the effects of testosterone on 
physical function measures: inclusion of healthy men with no functional limitations, 
confounding due to the ceiling and training effects, and the use of relatively low doses of 
testosterone which failed to significantly raise testosterone trials in some trials.   
In healthy older men, in whom endogenous testosterone production had been 
suppressed by administration of a GnRH agonist, graded doses of testosterone were 
associated with dose-dependent increments in appendicular skeletal muscle mass and 
leg press strength (92). Only limited data are available on the effects of testosterone 
therapy on mobility, as determined by walking speed. In a randomized clinical trial in 
which 70 men were randomized to receive testosterone enanthate intramuscularly with 
or without finasteride or to receive placebo for three years, the men treated with 
testosterone, in comparison to those who received placebo, experienced significantly 
greater increases in a composite timed test of physical function that included walking 
speed (139). In another study of patients with congestive heart failure, testosterone 
therapy was associated with greater increase in shuttle walk distance than placebo 
(150). In this study, the increments in walking distance were correlated with changes in 

 
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bio-T levels (150).  A randomized SARM trial also found significant improvements in 
stair climbing power (151).  Accordingly, we have emphasized measures of skeletal 
muscle mass, muscle performance, and physical function, which have been shown to 
be androgen responsive and which are important for activities of daily living and mobility 
(e.g., walking and stair climbing).   
3. 
Study Design 
This will be a randomized, placebo-controlled, parallel group, double blind, clinical trial 
in community dwelling, older men, 65 years of age or older, who have mobility limitation.  
The study will have a 2 X 2 factorial design, which will allow us to investigate the effects 
of dietary protein intake and testosterone separately and together. 
4. Outcome Measures 
4.1. 
Primary Outcome Measure 
Our primary outcome is change in lean body mass, measured by dual energy X-ray 
absorptiometry (DXA), because lean body mass is an excellent marker of whole body 
protein anabolism, is responsive to testosterone administration, and can be measured 
accurately and precisely by DXA (152-153). At a population level, sarcopenia defined in 
terms of lean body mass is predictive of disability, fracture risk, and other outcomes (34-
36, 154). We recognize that translation of lean body mass gains into muscle 
performance and physical function gains is crucial for establishing the efficacy of 
function promoting therapies. Accordingly, we have included measures of muscle 
performance and physical function. 
4.2. 
Secondary Outcome Measures 
Tests of Muscle Performance 
i. Maximal voluntary strength measured by the 1-repetition maximum method in the 
leg press exercise  
ii. In addition, we will measure maximal voluntary strength in the chest press exercise. 
This exercise was chosen because it involves the large muscle groups of the upper 
extremities. 
iii. We will measure the power of hip and knee extension by Bassey’s leg rig. 
Tests of Physical Function and Task-Specific Performance 
i. 6-minute walking distance and speed (which is a reciprocal of walking distance) 
ii. Stair-climbing power and speed without and with 20% load carry  
iii. 50-meter timed walk with 20% load carry 
d.  Self-Reported Physical Function. We will characterize self-reported physical 
function by using the physical function domain of the Medical Outcomes Study Short 
Form-36 (SF36). Systematic reviews of randomized trials have shown that testosterone 

 
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therapy improves self-reported physical function, as assessed by the physical function 
domain of SF-36.   
e. Sense of well being will be assessed by Psychological Well Being Index, and fatigue 
by FACIT-1 Fatigue scale; these outcomes have been shown to be androgen 
responsive. Positive and negative affect will be assessed by Derogatis Affective 
Balance Scale (DABS).     
Safety measures 
In accordance with the recommendations of the Endocrine Society’s Expert Panel for 
Testosterone Therapy of Androgen Deficiency Syndrome in Men (150), we will monitor 
the following to assure subject safety:  
i. Hemoglobin and hematocrit 
ii. Serum PSA levels, periodic digital rectal examinations of the prostate, and American 
Urological Association/ International Prostate Symptom Score (AUA/IPSS) score 
iii. Plasma lipids and apolipoprotein levels 
There is no evidence that testosterone enanthate, when administered by intramuscular 
injection at the proposed dose, affects liver enzymes. There is weak evidence linking 
testosterone therapy to obstructive sleep apnea; both worsening and improvements in 
sleep apnea have been reported with testosterone administration, leading the Endocrine 
Society expert panel not to recommend monitoring for sleep apnea. Although there is no 
evidence that protein intakes of up to 2 g• kg-1• day-1are associated with adverse health 
effects, we will monitor: 
i.  24-hour urinary calcium excretion 
ii. Blood urea nitrogen (BUN) and creatinine 
iii. Blood chemistry panel, including albumin, prealbumin and transferrin as markers 
of protein status 
This study is neither sufficiently long in duration nor is adequately powered to determine 
the effects on bone mineral density and fracture risk. Demonstration of the beneficial 
effects on muscle mass, muscle performance, and physical function in this trial may 
justify a larger and longer study.    
5. 
Subject Selection and Withdrawal 
5.1. 
Number of Subjects 
We estimate that 152 men will be needed to test the primary hypothesis, based on 
assumption of two-sided, type I error rate of 0.05 and an intent-to-treat analytical 
strategy.  We assume that no more than 20% of subjects in four groups will drop out 
before end of study.  
5.2. 
Inclusion Criteria 
1. 
Community-dwelling men 65 years of age or older 

 
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2. 
A score of 3-10 on the short physical performance battery (SPPB). If score is 10, 
than combination must meet 4-3-3 (Balance, Walk, Chair stand) 
3. 
Daily protein intake less than the recommended daily allowance of 0.83 g • kg-1 • 
day-1 (from the average of three 24-hour food recalls or below 0.83 on two out of 
three days)   
4. 
Able to give informed consent 
4.3. 
Exclusion Criteria  
1) 
History of prostate or breast cancer  
2) 
American Urological Association [AUA] symptom index score of > 19 
3) 
Prostate specific antigen (PSA) > 3 ng/ml in Black men or > 4 ng/ml in men who 
are not Black (since PSA levels are inherently variable and show lot of 
fluctuation, PSA levels may be repeated once if the initial levels were close to our 
eligibility criterion)   
4) 
PSA > 3 ng/ml in those with history of prostate cancer in first degree relatives. 
These subjects may be enrolled if they have a negative transrectal biopsy within 
the past year. 
5) 
Myocardial infarction or stroke within the last 6 months 
6) 
Uncontrolled congestive heart failure, based on the study physician’s evaluation 
7) 
A. Serum creatinine > 2.0 mg/dL  
B. Men on any kind of dialysis will be excluded.  
8) 
History of celiac disease, Crohn’s disease, or ulcerative colitis  
9) 
History of any malignancy requiring treatment within the previous 2 years, except 
non-melanomic skin cancers. Men with cancers who have not required active 
treatment within the past two years and who have not had disease recurrence 
within the past two years may be enrolled at the discretion of the study physician.  
10) 
Neuromuscular diseases: motor neuron diseases, multiple sclerosis, adult 
muscular dystrophies, and myasthenia gravis 
11) 
History of stroke with residual limb weakness that affected the individual’s ability 
to walk; subjects with history of stroke who do not have residual limb weakness 
may be enrolled.   
12) 
Schizophrenia, bipolar disorder, or untreated diagnosed depression. Subjects 
with unipolar depression who are on an antidepressant medication are eligible. 
13) 
TSH levels < 0.4 or > 7.5 mlU/L(Since TSH levels are inherently variable and 
show lot of fluctuation, TSH levels may be repeated once if the initial levels were 
close to our eligibility criterion) 
14) 
Systolic blood pressure (BP) > 160 or diastolic BP > 100 mm Hg (average of 2 
measurements taken at visit 1) (Blood pressure may be repeated at visit 2 if the 
participant forgot to take antihypertensive medications at visit 1. Blood pressure 
may also be repeated at visit 2 or if the participant had elevated blood pressure 
at visit 1 but is interested in participating in the trial and wants to speak with his 
PCP regarding medication adjustment). 

 
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15) 
A. Hemoglobin A1c > 8.0% 
B. Subjects on insulin therapy will be excluded.  
Men with diabetes mellitus whose A1C is less than 8.0% or who are not taking 
insulin will be eligible. 
16) 
Mini-Mental Status Exam [MMSE] <24 
17) 
Body mass index (BMI) less than 20 or greater than 40 kg/m2 
18) 
Vegans and strict vegetarians, who are not willing to eat red meat, fish, shellfish, 
poultry, or eggs   
19) 
Allergy to peanuts, soy, sesame, shellfish, or gluten 
20) 
Current alcohol use >21 drinks/week based on self-report 
21) 
Confinement to a wheelchair 
22) 
Current use of any of the following medications: 
a) 
Use of other anabolic therapies: testosterone, DHEA and androstendione, 
or rhGH within 1 year of screening 
b) 
levodopa 
23) 
Current enrollment in a structured weight management program or participation 
in any weight intervention studies in the preceding 90 days  
24) 
Serum ALT and AST greater than 3 x upper limit of normal 
25) 
Hematocrit < 30% or >48% (Since hematocrit values can transiently increase as 
a result of dehydration, and the subjects in this trial are required to come after an 
overnight fast, hematocrit levels may be repeated once if the investigators 
determine that the initial elevated hematocrit was influenced by dehydration) 
5.3. 
Subject Recruitment and Screening 
We will use a step process to streamline screening of subjects: telephone screening 
(step 1), in-person interview and blood tests for blood counts and chemistries, PSA and 
total and free testosterone, analysis of three 24-hour food recalls, and a Short Physical 
Performance Battery (step 2). Subjects who respond to our advertisements will undergo 
telephone screening that conforms to HIPAA guidelines and in which we will verify key 
inclusion and exclusion criteria by using a standardized questionnaire. Those who 
conform to the eligibility criteria during the phone screen will be invited to come to the 
BWH, where they will sign the consent form after a detailed explanation of the study, 
and undergo medical history, physical examination, blood counts and chemistries, and 
measurement of PSA. The subjects will also undergo a SPPB. Eligibility will be 
determined in two screening visits (screening visits SV1 and SV2) based on conformity 
to inclusion and exclusion criteria using structured interview, questionnaires, physical 
examination, blood tests, ECG, 24-hour food recalls, and an SPPB test. 
5.4. 
Early Withdrawal of Subjects 
Because these trials are based on the principle of “intent-to-treat”, this requires that to 
the degree possible, all subjects randomized are followed to obtain outcomes data, 

 
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regardless of whether they complete their intervention regimens or are noncompliant.  
This preserves the effect of randomization.  Therefore, ideally, a subject would be 
followed for all outcomes even in the case where he has elected cessation of study 
intervention. 
Therefore the study design explicitly differentiates between cessation of study 
intervention and withdrawal from study.  The subject may elect either at any time.  The 
former may also be elected by the study physician (due for instance to safety or 
compliance concerns as explained above), but we anticipate that the study physician 
would invoke the latter quite rarely, if at all.  
If a subject decides to cease study intervention, this will be recorded on Weekly 
Participant Status Form and he will no longer receive the intervention to which he has 
been randomized.  Under this scenario study visits would continue as scheduled and 
the subject would continue to be followed for all endpoints (including adverse events, 
physical performance testing, etc.)  This scenario would also apply if the study physician 
elects to stop intervention. 
If by contrast the subject elects full withdrawal from study, this would be recorded on 
Participant Final Disposition Form and all contact with the subject would cease.  If 
the request for study withdrawal is not communicated in person (i.e. the request is 
received over the phone), staff will request a final in-person visit with the subject to 
complete a final safety evaluation prior to cessation of contact with subject.  The subject 
may obviously refuse this request but past experience indicates that subjects are 
usually willing to be seen for a final disposition visit. 
5.5. 
Restrictions during the course of the study 
The participants will be advised that their participation in the study is contingent upon an 
agreement not to: 
 
Engage in progressive and heavy weight lifting exercise during the course of the 
study. The subjects are allowed to continue mild to moderate endurance exercise 
provided they agree not to change the intensity of the exercise. 
 
Participate in a structured weight loss program 
 
Start a nutritional supplement other than that provided by the study staff during 
the course of the study 

 
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6. 
Study Drug  
6.1. 
Description 
The study drug is testosterone enanthate (Delatestryl, ENDO Pharmaceuticals).   
6.2. 
Treatment Regimen 
The subjects will receive either placebo or testosterone enanthate 100 mg 
intramuscularly weekly for six months. All injections will be administered in the GCRU or 
the study clinic at BWH.  
6.3. 
Drug Storage 
Bulk supplies of study medication will be stored in the IDS Pharmacy at controlled room 
temperature (20-25 Celsius).  Study medication will be labeled by the IDS Pharmacy for 
individual study subjects.   
6.4. 
Subject Randomization and Stratification 
Subjects who meet the eligibility criteria will be randomly assigned to one of four 
treatment groups based on a stratified randomization schedule developed by the 
biostatistician, using the permuted blocks strategy with randomly varying blocks of size 
4 and 8.  Stratification will be by diabetes status. 
Group A:  
Placebo injections weekly; 0.8 g/kg/day protein 
Group B:  
Placebo injections weekly; 1.3 g/kg/day protein 
Group C:  
Testosterone enanthate 100 mg intramuscularly weekly; 0.8 g • kg • 
day protein 
Group D:  
Testosterone enanthate 100 mg intramuscularly weekly; 1.3 g • kg • 
day protein 
6.5.  Blinding 
The subjects, the research coordinators, and the personnel involved in outcomes 
assessments and monitoring compliance will all be blinded. Treatment assignment will 
be known only to the Data Management Team and the Investigational Drug Pharmacy. 
The testosterone enanthate injections and the sesame oil placebo injections will have 
identical appearance and consistency. An unblinded physician will be designated to 
monitor safety results. All participants will receive packaged meals plus a supplement to 
ensure blinding. The dietary supplements containing either casein and whey mix or 
carbohydrate and fat mix will have identical appearance.   

 
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7. 
Study Procedures and Visits 
7.1. 
Telephone Screening  
Potential subjects who call the trial site in response to advertisements or letters will be 
asked a set of structured questions to verify key eligibility criteria.  
If a potential subject is willing to answer questions, is ≥65 years old and meets the 
inclusion and exclusion criteria that are assessed during the telephone interview, he will 
be asked to schedule Visit 1, the first in-person visit.    
7.1.1. 
Visit 1 (V1) 
Subjects will first be asked to give written, informed consent.   
Visit 1 - Assessments and Procedures 
 Screening Consent 
 Medical history  
 Medications 
 Questionnaires: AUA/IPSS  
 Height and weight (for BMI)  
 Blood pressure 
 MMSE 
 SPPB 
 24-hour food recall 
 
At V1, The study dietitian will perform a 24-hour food recall and instruct the subjects on 
how two additional 24-hour recalls will be conducted. For all subjects meeting the 
eligibility criteria assessed at V1, two additional 24-hour recalls will be completed on 
randomly assigned days between V1 and V2. The subjects, who meet the eligibility 
criteria for protein consumption, as well as all other criteria assessed during V1, will be 
asked to return for V2.  
 
Blood pressure may be repeated at visit 2 if the participant forgot to take 
antihypertensive medications at visit 1. Blood pressure may also be repeated at visit 2 
or if the participant had elevated blood pressure at visit 1 but is interested in 
participating in the trial and wants to speak with his PCP regarding medication 
adjustment.  
7.1.2. 
Visit 2 (V2)   
The following procedures and questionnaires will be completed:  
 Physical examination, including a digital rectal examination (DRE) 
 Blood draw for CBC, chemistry panel, PSA, A1C 
 ECG 
 Rapid assessment of physical activity form (RAPA) 
 
24-hour food recall 

 
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 Body weight 
 
Adverse event and concomitant medication will be reviewed 
 
Eligibility will be determined at the completion of V2 and after the results of the tests 
performed during V2 become available. Subjects who meet ALL the inclusion criteria 
and NONE of the exclusion criteria will be scheduled for enrollment in the Run-in 
Period.  
 
Subjects may be asked to return for a repeat PSA, TSH or Hematocrit (rationale 
elaborated on pages 15 and 16). 
7.1.3. 
The Run-in-period 
During the run-in-period, the subjects will be provided a diet containing 0.8g/kg/day 
protein for a duration of 10 to 16 days.  Body weight will be recorded weekly.  Adverse 
event and concomitant medication will be reviewed during this period.  Subjects who 
complete the run-in-period successfully will be eligible for randomization.  Subjects may 
be excluded at the end of the Run-in Period on the basis of the following: (a) failure to 
return for the baseline study visit, (b) failure to consume at least 75% of the meals 
provided for reasons of palatability or tolerability, (c) taste aversion to supplements, or 
(d) any other reason cited by the study investigator or medical team that would interfere 
with the subject’s completion of the 6-month trial. The investigator may exclude subjects 
even if they meet all the listed eligibility if the investigator determines that participation in 
the study may be harmful to the participant’s health, or if the investigators feel that the 
subject may not be able to comply with the protocol for any reason.  
If a subject meets all eligibility criteria except the 75% compliance with the meals, due to 
intolerance of or dislike of food items in the study meals, they will be offered a modified 
meal plan. The modified (or hybrid) meal plan will allow subjects to replace either lunch, 
dinner or both meals with meals cooked by the subjects themselves. The study dietitian 
will instruct these subjects on how to prepare their own meals based on their caloric and 
protein goals. The run-in period of subjects requiring the modified meal plan may be 
extended by approximately one week. Subjects who are determined to be compliant 
after the hybrid-run-in phase will be eligible for randomization.  
 
Baseline assessments will be completed for all subjects during or at the end of the run-
in period after the eligibility has been determined.  
7.1.4. 
Baseline Visit 
After full consent is obtained, the following baseline studies and procedures will be 
performed: 
 Blood tests:  
o Blood counts 
o Blood chemistries, including ALT, AST, bilirubin and alkaline phosphatase, 
BUN, creatinine, prealbumin, transferrin, and albumin 
o Plasma lipids and apolipoproteins  

 
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o Serum PSA 
o Two ten ml blood samples drawn ten minutes apart and pooled for the 
measurement of total and free testosterone, DHT, estradiol, LH, and 
SHBG. 
 ECG 
 Anthropometrics. Weight (same scale, minimal clothing) will be recorded.   
 Body Composition by a whole body DXA scan 
 Muscle performance (maximal voluntary strength, leg power) 
 Physical function (6-minute walk, loaded and unloaded stair climbing power and 
speed, load carrying walk test) 
 Questionnaires: SF-36, Psychological Wellbeing Index, DABS, the FACIT-1, and 
RAPA 
 24-hour urine collection for calcium, total nitrogen and creatinine 
 Structured assessment of cardiovascular events 
 
If the subject is completing a second run-in period on the hybrid meal plan, evaluation of 
meal compliance will take place prior to consenting for the full study. 
7.2. 
Randomization 
The subjects will be randomized at the completion of the baseline assessments. The 
day of randomization will be called day 0.    
7.3. 
Intervention Period 
The subjects will be scheduled for weekly injections of study medication at the time of 
their randomization. Body weight will be recorded weekly. Adverse events and 
concomitant medications will be reviewed at each visit.  Subjects will return to the study 
clinic each week to receive intramuscular injection of their study medication. 
 
Day 0, Day of Randomization (Visit 6) 
 Randomization 
 Injection of study medication 
 Dietary instructions 
 Dispense food and study supplement 
 Dispense vitamin and calcium supplement 
 
Week 1 (Visit 7) 
 Injection of study medication 
 Dispense food 
 Dietary instructions 
 Dietary checklist 
 

 
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Week 2 (Visit 8) 
 Injection of study medication 
 Dispense food and study supplement 
 Structured evaluation of cardiovascular events 
 Vital signs 
 24-hour food recall to verify dietary compliance 
 
Week 3 (Visit 9) 
 Injection of study medication 
 Dispense food 
 Dietary checklist 
 
Week 4 (Visit 10) 
 Injection of study medication 
 Vital signs 
 Dispense food and study supplement 
 Dispense vitamin and calcium supplement 
 24-hour food recall for dietary compliance 
 Optional RAPA for diet level adjustment 
 
Week 5 (Visit 11) 
 Injection of study medication 
 Dispense food 
 Dietary checklist 
 
Week 6 (Visit 12) 
 Injection of study medication 
 Physical examination, including a digital prostate examination 
 Blood test for CBC, chemistry panel, and hormones 
 Urinary symptom assessment with AUA/IPSS 
 Dispense food and study supplement  
 24-hour food recall for dietary compliance 
 
Week 7 (Visit 13) 
 Injection of study medication 
 Dispense food 
 Dietary checklist 
 
Week 8 (Visit 14) 
 Injection of study medication 
 Dispense food and study supplement 
 Dispense vitamin and calcium supplement 
 24-hour food recall for dietary compliance 
 

 
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Week 9 (Visit 15) 
 Injection of study medication 
 Dispense food 
 Dietary checklist 
 
Week 10 (Visit 16) 
 Injection of study medication 
 Dispense food and study supplement 
 24-hour food recall for dietary compliance 
 
Week 11 (Visit 17) 
 Injection of study medication 
 Dispense food 
 Dietary checklist 
 
Week 12 (Visit 18) 
 Injection of study medication 
 Physical examination, including digital prostate examination 
 Blood test for CBC, chemistry panel, PSA, and hormones 
 ECG 
 Urinary symptom assessment with AUA/IPSS 
 Structured evaluation of cardiovascular events and other adverse events 
 Dispense food and study supplement 
 Dispense vitamin and calcium supplement 
 24-hour food recall for dietary compliance 
 DXA  
 Muscle performance and physical function assessment 
 Questionnaires: SF-36, AUA/IPSS, Psychological Wellbeing Index, DABS, the 
FACIT-1, and RAPA 
 Optional diet adjustment based on RAPA score 
 24-hour urine collection for calcium, total nitrogen and creatinine 
 
Week 13 (Visit 19) 
 Injection of study medication 
 Dispense food 
 Muscle performance and physical function assessment 
 Dietary checklist 
 
Week 14 (Visit 20) 
 Injection of study medication 
 Vital signs 
 Dispense food and study supplement 
 24-hour food recall for dietary compliance 
 

 
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Week 15 (Visit 21) 
 Injection of study medication 
 Dispense food 
 Dietary checklist 
 Adverse event recording 
 
Week 16 (Visit 22) 
 Injection of study medication 
 Vital signs 
 Dispense food and study supplement 
 Dispense vitamin and calcium supplement 
 24-hour food recall for dietary compliance 
 
Week 17 (Visit 23) 
 Injection of study medication 
 Dispense food 
 Dietary checklist 
 
Week 18 (Visit 24) 
 Injection of study medication 
 Vital signs 
 Dispense food and study supplement 
 24-hour food recall for dietary compliance 
 Urinary symptom assessment with AUA/IPSS 
 Physical examination, including a digital prostate examination 
 Blood test for CBC, chemistry panel, and hormones 
 
Week 19 (Visit 25) 
 Injection of study medication 
 Dispense food 
 Dietary checklist 
 
Week 20 (Visit 26) 
 Injection of study medication 
 Vital signs 
 Dispense food and study supplement 
 Dispense vitamin and calcium supplement 
 24-hour food recall for dietary compliance 
 
Week 21 (Visit 27) 
 Injection of study medication 
 Dispense food 
 Dietary checklist 
 

 
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Week 22 (Visit 28) 
 Injection of study medication 
 Vital signs 
 Dispense food and study supplement 
 24-hour food recall for dietary compliance 
 
Week 23 (Visit 29) 
 Injection of study medication 
 Dispense food 
 Dietary checklist 
 
Week 24 (Visit 30) 
 Injection of study medication 
 Physical examination, including a digital prostate examination 
 Blood test for CBC, chemistry panel, PSA, and hormones 
 ECG 
 Structured evaluation of cardiovascular events 
 Dispense food and study supplement 
 Dispense vitamin and calcium supplement 
 24-hour food recall for dietary compliance 
 DXA  
 Muscle performance and physical function assessment 
 Questionnaires: SF-36, AUA/IPSS, Psychological Wellbeing Index, DABS, the 
FACIT-1, and RAPA 
 Urinary symptom assessment with AUA/IPSS 
 24-hour urine for calcium, total nitrogen, and creatinine 
 
Week 25 (Visit 31) 
 Muscle performance and physical function assessment 
7.4. 
Subject Compensation 
Subjects will be compensated during the course of the trial, based on the number of 
visits completed. In addition, parking and meal tickets will be provided for study visits.   
8. 
Dietary Prescription, and Enhancing and Verifying Dietary Compliance 
8.1. 
Standardizing energy and protein content through packaged meals and 
supplement 
Each subject’s daily energy requirements will be calculated using the Dietary Reference 
Intake equation plus an activity factor (Rapid Assessment of Physical Activity; RAPA); 
this method has been validated in controlled feeding studies (181). Two levels of protein 
intake will be used: 0.8 g • kg-1• day-1 (reference or control group) or 1.3 g • kg-1 • day-1 
(intervention group). The daily energy and protein allowances will be apportioned 

 
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between the study meals (pre-packaged or subject-prepared), the protein or energy 
(placebo) supplements, and a discretionary allowance. We will provide 80% of daily 
energy allowance through the packaged meals, leaving 20% of energy to be derived 
from the dietary supplements and discretionary calories. This approach offers flexibility 
and convenience and increases the likelihood of dietary adherence and maintenance of 
consistent portion control. Some subjects will be allowed to replace the study-provided 
meal (lunch and dinner only) with a meal they prepare themselves. Replacements will 
not be allowed for breakfast or the nutritional supplement. Subjects who are preparing 
their own lunch or dinner will be taught how to match the daily energy allowance amount 
as calculated by the Dietary Reference Intake equation plus an activity factor. 
Personal Chefs to Go, Inc. will provide meals with only five graded levels of protein and 
caloric content.  Thus, it is likely that the protein and energy content of the packaged 
meal will not match the estimated protein or energy requirement. The difference 
between the estimated protein and energy requirement per protocol and the protein and 
energy content of packaged meal will be made up by a protein and/or carbohydrate 
supplement.  These meals will provide 0.7 g • kg-1• day-1 of protein for subjects in the 
intervention and control groups. Calories in the packaged meals will be apportioned 
such that 20% of total calories are provided as breakfast, 35% as lunch, and 45% as 
dinner based on typical distribution of calories across meals in older participants of the 
Framingham Heart Study. Subjects who are preparing their own lunch or dinner will be 
taught how to match the total calories and protein content of the packaged meals. 
Subjects in the control group will receive all of their daily protein allotment (0.8 g • kg-1• 
day-1) through packaged meals provided by Personal Chefs To Go, Inc., supplement 
and discretionary calories (snacks). Subjects in the intervention arm will receive an 
additional supplement containing 0.5 g • kg-1• day-1 of a casein and whey protein mix, 
bringing their daily protein intake to 1.3 g • kg-1• day-1. To ensure comparability between 
the two groups, the control group will receive an additional supplement containing 0.5 g 
• kg-1• day-1 of a carbohydrate and fat mix (matched to the protein supplement in taste, 
texture and caloric content). The supplements will be divided into three portions to be 
ingested at breakfast, 3-4 PM (with a snack), and at bedtime. Subjects who are 
preparing their own lunch or dinner will be taught how to match the total calories and 
protein content of the packaged meals. 
To allow for some variety and subject choice, 15% of daily calories will be allocated as 
discretionary calories and will include such items as fruits and vegetables, coffee and 
tea with milk and/or sugar, alcoholic beverages, and other foods. The participants will 
record all discretionary items consumed on the Food Compliance Checklist provided 
with the packaged meals; Compliance Checklists will be collected every other week at 
the clinic visit.  
The table below illustrates how calories and protein may be distributed in the dietary 
prescription of a 70-kg person assigned to either the control (0.8 g • kg-1• day-1) or 
intervention (1.3 g • kg-1• day-1) group and assumed to have daily energy requirement of 
2100 Kcal is shown in the table below: 
 
Table 8.  
Energy and Protein Distribution in a 70-kg Man Assigned to either 0.8 or 1.3 g • kg-1• day-1 
Groups with estimated daily energy requirement of 2100 kcal 
Group 
assignment 
Energy in 
packaged 
Protein in 
pkg’d 
Energy in  
Supplement 
Protein in 
Supplement 
Discret. 
Calories 
Discret. 
protein  
Total 
energy 
Total 
protein 

 
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meals 
meals 
0.8 g/kg/day 
1600 kcal 
45 g 
185 kcal 
4 
315 kcal 
7 g 
2100 kcal 
56 g 
1.3 g/kg/day 
1600 kcal 
45 g 
185 kcal 
39 g 
315 kcal 
7 g 
2100 kcal 
91 g 
 
Each participant will receive instructions explaining how to apportion, store, and eat the 
packaged meals and supplements throughout the day. They will also be instructed in the types 
and amounts of discretionary foods they can eat to add variety. The men assigned to the low 
and high protein groups will receive identical packaged meals and supplement will be designed 
to look and taste alike. The importance of dietary compliance will be reinforced every week. The 
subjects will be provided a seven day supply of packaged food each time. They will pick up their 
weekly supply of food during their weekly visits to the study clinic or the meals will be shipped to 
their homes. 
Vitamins, minerals and micronutrients 
To assure that subjects receive sufficient amounts of vitamins and minerals, they will receive 
one generic multivitamin tablet, and a generic calcium citrate tablet containing 630 mg of 
elemental calcium twice daily and 500 IU of vitamin D (the RDA).  
Preparation of the Dietary Supplements 
The dietary supplements will be prepared by the manufacturers. For the intervention group 
received the 0.5 g • kg-1• day-1 protein supplement, the supplement itself will be prepared by 
mixing Milk Protein Concentrate-80 (MPC-80) with Whey Protein Concentrate-80 (WPC-80). 
The 80:20 casein to whey blend of MPC-80 will be diluted to a 50:50 casein:whey blend (CWB 
50:50) using an appropriate amount of WPC-80. The final CWB 50:50 will be unflavored to 
provide the greatest flexibility to the subjects for mixing and consuming the supplements at 
home. However, the protein supplement will be sweetened to match the taste of the 
carbohydrate (glucose-based) supplement. Quality control over the blend of casein to whey as 
well as the total protein concentration per unit of powder will be carried out for each batch at the 
manufacturing plant and results of the quality control analysis will be provided to the 
investigators with the shipment of each new batch.  
The protein and carbohydrate supplements will be delivered to the study dieticians in tubs for 
the purpose of individual packaging for study subjects. Supplements will be packaged in 
individual portions with the name, ID number and day and date for consumption. Color coding 
will be used to separate morning, afternoon and evening supplement doses. 
8.3. 
Compliance with Dietary Prescription 
We have incorporated several approaches to enhance dietary compliance, including the 
provision of packaged meals and portion control, the use of compliance checklists to assess 
compliance on an every two-week basis, and the use of this information in real time to reinforce 
dietary instructions every other week.  
In this study, subjects are provided pre-packaged frozen meals and protein/placebo 
supplements. Some subjects will be allowed to replace the study-provided meal (lunch and 
dinner only) with a meal they prepare themselves. Replacements will not be allowed for 
breakfast or the nutritional supplement. Subjects who are preparing their own lunch or dinner 
will be taught how to match the daily calories and protein content as calculated by the Dietary 
Reference Intake equation plus an activity factor. They will also be given recommendations to 
not eat out more than one meal per week.  
For subjects with lactose intolerance, the study dietician devises a specific meal plan to 
minimize dairy in the meals. However; as dairy is part of many food products and not completely 
avoidable, some subjects could experience gastrointestinal symptoms while on study meals.  In 

 
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order to improve compliance in these subjects we will prescribe lactase supplements to 
symptomatic subjects. The lactase supplement would be packaged by our IDS Pharmacy 
following the same procedures that are in place for the dispensing of multivitamin and 
calcium/vitamin D supplements for this study.  
Compliance with dietary prescription will be assessed by using alternating Compliance 
Checklists and 24-hour food recalls during alternating weeks. The subjects will return their 
Compliance Checklist at every other weekly visit. Subjects who are preparing their own lunch or 
dinner will be asked to keep detailed food records for 2 week days and 2 weekend days during 
alternating weeks. These subjects will return their Compliance Checklist at every other weekly 
visit. These subjects will continue to receive 24-hour food recalls every other week. 
Due to the large respondent burden associated with returning empty meal containers and 
incompletely-consumed meals, especially in older subjects with mobility problems, we will not 
require the return of such items. Instead, we will use a Dietary Compliance Checklist, a method 
that has been used successfully in previous studies by these investigators. The Dietary 
Compliance Checklist is a natural choice for a feeding study because of its simplicity, objectivity, 
and precision. The checklist, included with each meal, will list each item that is included in the 
meal package and the respondent will simply check off all foods eaten. For those foods that are 
not completely eaten, the subject will indicate what portion was consumed (e.g., one-quarter or 
one-half). Subjects will return their Compliance Checklists every other weekly visit.  
The participants will be instructed on how to fill out Dietary Compliance Checklist and will be 
given handouts with examples of completed food records. At every other weekly visit, the study 
nutritionist will review the Dietary Compliance Checklist and inquire about difficulties associated 
with compliance, including the amount of food provided, palatability, problems in filling the 
Compliance Checklist, and factors affecting the ability to follow the prescribed diet or 
supplements. The compliance will be calculated as the percent of daily energy and protein 
eaten, using the NDS-R, and averaged across the treatment period to obtain an average 
compliance score for each subject. The biweekly 24-hour food recalls will be used as a cross-
check on the compliance estimates obtained from the Compliance Checklists. The compliance 
estimates will be used a covariate in secondary analyses. These methods are established 
approaches to monitor compliance in dietary intervention studies and based on more than 10 
years experience of Dr. Apovian. 
8.4. 
Exercise and Activity 
All participants will be asked to abstain from resistance and heavy endurance exercise. Those 
involved in weight lifting exercises will be enrolled only if they agree to discontinue weight lifting 
at least twelve weeks before the start of the study. Those involved in mild to moderate aerobic 
exercises on a regular basis may continue to do so provided the intensity of exercise is 
maintained constant throughout the study period. 
9. 
Statistical Plan 
9.1. 
Analytical Methods and Sample Size Estimations 
Statistical Analyses.  Primary analyses will follow the “intention-to-treat” (ITT) 
principle; i.e., individuals will be analyzed according to their assigned treatment group 
whether or not they remain on the study treatment. Every attempt will be made to follow 
and continue to evaluate all enrolled subjects. The ITT approach avoids bias if 
individuals who drop out of the two arms have different prognoses.  For safety 

 
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outcomes, however, all individuals exposed to testosterone, whether or not it was their 
assigned treatment, will be considered. No assumptions will be made as to the 
consistency of the rate of change in outcomes with time.  A secondary, ‘per-protocol,’ 
analysis will also be conducted for each outcome with noncompliant subjects excluded.    
All analyses will be adjusted for randomization and stratification factors. The subjects 
will be randomized in equal parts to one of two protein regimens (0.8 g/kg/d vs. 1.3 
g/kg/d), and within each of these to testosterone or placebo, so that four subcohorts 
(coded A-D in the table below) of equal size may be compared to one another to assess 
the marginal and joint influences of testosterone and enhanced protein intake. The 
primary outcome is lean body mass, measured by DXA, and the primary focus of the 
statistical analysis estimation and inference surrounding the effects of 1.3 g diet and 
testosterone supplementation on lean body mass above and beyond those attributable 
to 0.8 g protein and placebo, respectively. Leg press strength, chest press strength, leg 
power, walking speed, stair climbing power, load carrying speed, self-reported physical 
function, fatigue, well being and affectivity balance will be considered as secondary 
outcomes.  Based on extensive experience, we assume an overall rate of attrition of no 
more than 20% of subjects during the six-month trial period (see below).     
Descriptive and Exploratory Analyses.  Prior to formal modeling, exploratory 
methods will be employed to assess data quality, preliminary evidence of associations 
and to detect the presence of outlying values and potential influence points. Means, 
standard deviations, medians, and ranges will be computed for continuous variables, 
and sample probability mass functions computed for categorical factors. Histograms, 
scatter plots, and box plots will be used to assess skew and examine assumptions 
(such as normality) underlying statistical models. Baseline characteristics for the four 
randomized subcohorts will be compared using ANOVA for continuous variables and 
Fisher’s exact tests for categorical variables. Where appropriate, log or power 
transformations of outcome variables will be employed to enhance conformity with 
assumptions (e.g. normality in outcomes and/or linearity in associations between 
outcomes and covariates). Exploratory graphical assessment of the strength of 
association between covariates and outcome variables will be obtained via scatterplot 
smoothing using Generalized Additive Models. A preliminary assessment of the quantity 
and pattern of missing data will also be performed.  
Compliance Analysis. Compliance will be evaluated in two areas: dietary compliance 
and the number of injections. Dietary compliance will be assessed by means of 
alternating Dietary Compliance Checklist and 24-hour food recalls as described in 
another section. The percent of injections received at the weekly clinic visits will be 
calculated. The primary intent to treat analysis will include all subjects; we will also 
perform secondary analyses using compliers. 
Hypothesis Testing. The primary analysis of lean body mass will be performed in two 
parts. Our primary analytical strategy is to use mixed-effects regression analysis to 
assess 3 and 6 month outcomes simultaneously with baseline total body lean mass 
considered as a covariate, as is appropriate for between-group comparisons of 
randomized subcohorts, and 6-month differences between arms will be estimated via a 
treatment contrast and corresponding 95% confidence interval. We will also use a two-
way ANOVA to compare change from baseline to 6-month across the four randomized 

 
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subcohorts. We expect general agreement between estimates arising from Parts I and 
II, but owing to its use of all data, that from mixed effects regression analysis will be 
considered the principal estimate of treatment effect.  Though the primary aim of the 
analysis will be to estimate treatment effects at 6-months, a linear trend in change with 
time will not be assumed, and the potential for differential mean trajectories in treatment 
effects over the entire duration of follow-up will be assessed via stratification and the 
use of statistical interaction terms.  Unless otherwise noted, all comparisons will assume 
an allowed type-I error probability of 0.05. 
Hypothesis 1 is concerned with the effect of 1.3 g • kg-1 • day-1 protein administration 
on body composition, strength and function as compared to the effects of RDA (0.8 g • 
kg-1 • day-1).  The effect of enhanced protein intake will be tested using a mixed effects 
regression model on all subjects, comparing those with enhanced protein to RDA 
controlling for administration (or lack of administration) of testosterone as well as 
stratification factors and baseline outcome measures (i.e. an ANCOVA scheme). 
Modification of the protein effect by testosterone administration will be investigated by 
the use of statistical interaction terms, which will be considered significant if achieving 
the 0.10 threshold.        
Hypothesis 2 is concerned with the differential effect of testosterone administration in 
the enhanced protein administration arms vs the RDA arms.  Again a mixed effects 
regression analysis will be performed, restricted to subjects receiving testosterone. 
Comparison of baseline to follow-up change in outcomes will then be performed 
between the protein subcohorts (1.3 g / k/day vs 0.8 g/k/day), controlling for stratification 
factors.   Analyses of secondary outcomes will proceed according to the same program. 
As secondary, exploratory analyses, we are also interested in knowing whether 
changes in lower extremity lean mass (measured by DEXA) correlate with changes in 
leg press strength and physical function. For these variables, we will generate the 
Pearson correlation coefficients. Appropriate scatter plots will be used to assess 
assumptions of linearity and homoscedasticity. If necessary, Spearman correlations will 
be computed to confirm the main results. We will also fit regression models on the 
change in leg press strength with terms for group, baseline protein intake, baseline 
testosterone levels, and the group by baseline testosterone interaction to assess if 
treatment works to a greater or lesser extent in men with low testosterone or low protein 
intake.  
We will examine whether changes in muscle strength correlate with changes in the 
measures of physical function (6-minute walking speed and time, stair climbing power), 
and self-reported physical function. If testosterone treatment does improve muscle 
strength, and also improves physical function and self-reported physical function, it 
would be of interest to know whether changes in muscle strength correlate with changes 
in self-reported and objective measures of physical function. 
Missing data.  Primary analyses will employ mixed-effects regression in order to utilize 
all available data under the ITT framework. No assumptions will be made concerning 
data lost to attrition (e.g. crude imputation techniques such as last-observation-carried-
forward will not be employed).  No technique can perfectly assess the precise influence 
of missing data. We will therefore employ sensitivity analyses to assess the potential 

 
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influence of missingness on conclusions, as is advocated by numerous authors; 
moreover, outcomes data collection will continue regardless of compliance with 
randomly assigned regimens, thus minimizing the influence of missingness under ITT.  
For instance we will compare baseline outcome and covariate values for all subjects 
with missing outcomes vis-a-vis those with complete data.  A lack of systematic 
differences is consistent with but does not prove the robustness of conclusions to bias 
from the missing data process. The conclusions of these sensitivity analyses will be 
incorporated in published reports of findings from the proposed study. 
Multiple Comparisons: We recognize that a number of hypotheses will be tested, 
which will increase the likelihood of committing a type 1 error. We are aware of many 
different approaches that can be used to adjust for multiple comparisons.  A two -sided, 
5% level of significance will be used for each pre-specified hypothesis and magnitude 
of effect size will be carefully evaluated for clinical significance.  For exploratory 
hypotheses, we will adjust the p value by using the Boneferroni’s correction. It is still 
possible that we will find statistical significance in some instances when the differences 
are of little clinical significance.  In such cases, the clinical importance of findings – 
whether the magnitude of treatment effect meets or exceeds the minimal clinically 
important difference in the outcome - will be highlighted in the interpretation of results.   
b.  Sample Size Estimate (Approved by the DSMB in its June 2016 meeting) 
 
We estimate that 92 men will be needed to test the primary hypothesis. We base this 
estimate on the following considerations: 
 Two-sided, type I error rate of 0.05.   
 
 We assume that the groups will be balanced at baseline due to successful 
randomization. 
 
 The primary analysis will use an intent-to-treat strategy in which all will be analyzed 
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 Baseline calculations below apply to Part A of the primary analysis, which is 
restricted to comparisons at 6 months controlling for baseline outcomes 
measurements using a linear regression model with estimates of both active 
treatment effects simultaneously. Part B (mixed effects modeling) will achieve 
greater power than is claimed below.  
 Additive intervention effects. The potential for synergistic influence of interventions 
will be considered in exploratory analyses.  
 We assume that no more than 15% of subjects in four groups will drop out before 
end of study. The dropout rate in our previous testosterone dose response study in 
older men which had rigorous dietary requirements was 12%.  
 
Primary comparisons.  This study will be powered to detect clinically meaningful 
differences in within-subject changes in lean body mass attributable to randomization to 
testosterone therapy or 1.3 g protein diet above and beyond those attributable to 
placebo and 0.8 g protein diet, respectively. Interaction between the interventions will be 
addressed in exploratory analyses, but will not be the primary focus of these 
computations. 
 
The expected pattern of gain in lean body mass among the four randomization groups 
(A, B, C, D) is depicted in the table below.  We anticipate that the group of subjects 
receiving placebo treatment and the 0.8 g protein diet will experience some marginal 
gain in lean body mass, denoted μ.  Subjects receiving the elevated protein diet and / or 
testosterone will exhibit greater gains, with the apparent effects of testosterone and 1.3 
g diet exhibiting an additive relation.  The anticipated mean ± SD gain in lean body 
mass attributable to testosterone therapy in this design (denoted in the table as the 
difference between cohorts A and C, or B and D) is expected to be approximately 2.8 kg 
with standard deviation no greater than 4.2 kg - yielding an effect size of at least 0.67 – 
while the gain attributable to 1.3 g diet is anticipated to be approximately 1.3 kg with SD 
about 2.3 kg, yielding an effect size of approximately 0.65.  These estimates are 
reasonable and conservative because in our testosterone dose response study, older 
men receiving the same dose of testosterone and 1.2 g protein• kg • day gained 4.3 kg 
(SD 2.3 kg, Preliminary Data) lean body mass.   
 
If the hypothesized differences hold, a sample size of 19 participants in each of the four 
treatment groups (total sample size = 76 men) would provide 80% power to detect these 
effects, while group-specific evaluable sample sizes of 26 and 29 would provide 
approximately 85% and 90% power to detect these effects, respectively. To insure 80% 
power for primary comparisons we therefore propose an enrollment sample of 76 / 0.85, 
i.e. 92 participants or 23 per cell.  
 
Although based on previous studies, we have hypothesized that there will no significant 
differences in change in lean body mass in older individuals receiving 0.8 g protein or 
1.3 g protein, the proposed sample size will be able to detect a difference of 1.3 kg in 

 
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the change in lean body mass between these two groups (SD 2.3 kg), which we posit is 
clinically meaningful. 
Secondary comparisons.  The anticipated analytic sample size of 92 subjects 
provides sufficient power to detect clinically meaningful changes in most of the 
secondary outcomes with 80% power. We anticipate, for instance, that the change in 
leg press strength in testosterone plus 0.8 g protein group will be 15 kg (SD 24 kg) and 
in testosterone plus 1.3 g protein group will be 24 kg (SD 30 Kg). This assumption is 
conservative based on previous testosterone trial which revealed an increase of 28 kg 
in leg press strength (SD 22 kg). We anticipate therefore that the true effect size of the  
 
testosterone effect on leg press strength will be approximately 0.56, detectable with 
80% power under the proposed design.  We assert that a 15 kg increase in leg press 
strength is clinically significant based on the fact that Fiatarone et al demonstrated that 
a 10 kg increase in leg press strength was associated with significant improvements in 
physical function, as assessed by stair climbing power, walking speed, and time for sit-
to-stand transition.  The proposed sample size will allow us to detect a difference of 10 
points in physical function domain of SF-36 HRQOL instrument in testosterone and 
placebo-treated men and a difference of 15 points between testosterone plus 1.3 g 
protein and placebo plus 0.8 g protein with >80% power. These differences are clinically 
meaningful as Ware et al have deemed a change of 8 point in the physical function 
domain of SF-36 to be clinically meaningful. The proposed sample size will allow us to 
detect this effect size with 80% power.  
9.2. 
Adverse Events  
We will compare proportions of men experiencing adverse events in each treatment 
group, with particular attention to areas that are plausibly associated with testosterone, 
including erythrocytosis, urinary tract symptoms, and prostate-related events or to 
protein intake such as kidney stones or hypercalciurea.  
9.3. 
Interim monitoring   
Once in progress, the study will be monitored by the Data and Safety Monitoring Board 
(DSMB), an independent group that will periodically review the results in order to assess 
safety. No interim efficacy assessment is planned. The DSMB will receive semi-annual 
Table 1. Anticipated mean change in lean body mass according to protein diet and assignment to 
testosterone or placebo therapy.  The baseline expected increase μ present in the placebo + 0.8 g 
reference cell (upper left) will be augmented in the other subcohorts by the additive effect of 
additional protein intake and/or Testosterone therapy. 
 
Protein Regimen 
Placebo 
T 
0.8 g / d 
A: μ 
C: μ + 2.8 kg 
1.3 g / d 
 B: μ + 1.5 kg 
D: μ + 4.3 kg 

 
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reports and will meet annually, The DSMB will decide if an early stopping rule would be 
used to recommend termination of the study. 
10. Safety and Adverse Events 
10.1. 
Definitions 
Definitions are per the January 2007 Guidance on Reviewing and Reporting 
Unanticipated Problems Involving Risks to Subjects or Others and Adverse Events, 
Office on Human Research Protection (OHRP) Guidance. 
http://www.hhs.gov/ohrp/policy/AdvEvntGuid.htm. The requirements and processes for 
reporting adverse events are described in the corresponding National Institute on Aging 
(NIA) Guidelines.  
10.1.1. 
Adverse Event  
An adverse event (AE) is any untoward or unfavorable medical occurrence in a human 
study participant, including any abnormal sign (for example, abnormal physical exam or 
laboratory finding), symptom, or disease, temporally associated with the participant’s 
involvement in the research, whether or not considered related to the subject’s 
participation in the research.  
10.1.2. 
Serious Adverse Event  
A serious adverse event (SAE) is any AE that is:  
 fatal 
 life-threatening 
 requires or prolongs hospital stay 
 results in persistent or significant disability or incapacity 
 results in congenital anomalies or birth defects 
 an important medical event* 
Important medical events* are those that may not be immediately life threatening, but 
are clearly of major clinical significance.   
10.1.3. 
Unanticipated Problem 
An Unanticipated Problem is any incident, experience, or outcome that meets all of the 
following criteria: 
 unexpected (in terms of nature, severity, or frequency) given the research 
procedures that are described in the IRB-approved research protocol and 
informed consent document; 
 related or possibly related to participation in the research; possible related means 
that there is a reasonable possibility that the incident, experience or outcome 
may have been caused by the procedures involved in the research.  

 
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 suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) related to the 
research than was previously known or recognized. 
10.1.4. 
Adverse Event Reporting Period  
The study period during which adverse events must be reported is normally defined as 
the period from the initiation of any study procedures to the end of the study treatment 
follow-up. 
10.1.5. 
Preexisting Condition  
A preexisting condition is one that is present at the time of signing the consent form for 
the main study.  A preexisting condition should be recorded as an adverse event if the 
frequency, intensity, or the character of the condition worsens during the study period. 
10.1.6. 
General Physical Examination Findings  
At screening, any clinically significant abnormality should be recorded as a preexisting 
condition.  At the end of the study, any new clinically significant findings/abnormalities 
that meet the definition of an adverse event must also be recorded and documented as 
an adverse event.  
10.1.7. 
Post-study Adverse Event  
All unresolved adverse events should be followed by the investigator until the events 
are resolved, the subject is lost to follow-up, or the adverse event is otherwise 
explained.  At the last scheduled visit, the investigator should instruct each subject to 
report any subsequent event(s) that the subject, or the subject’s personal physician, 
believes might reasonably be related to participation in this study.  The investigator 
should notify the study DCC of any death or adverse event occurring during the year 
after a subject has completed treatment.  
10.1.8. 
Abnormal Laboratory Values  
A clinical laboratory abnormality should be documented as an adverse event if any one 
of the following conditions is met:  
 The laboratory abnormality is not otherwise refuted by a repeat test to confirm 
the abnormality 
 The abnormality suggests a disease and/or organ toxicity 
 The abnormality is of a degree that requires active management 
10.1.9. 
Hospitalization, Prolonged Hospitalization or Surgery  
Any adverse event that results in hospitalization should be documented and reported as 
a serious adverse event.  Any condition responsible for surgery should be documented 
as an adverse event if the condition meets the criteria for an adverse event and 
reported as a severe adverse event if hospitalization is required.  Neither the condition, 
hospitalization, nor surgery are reported as an adverse event if the hospitalization was 
for diagnostic or elective surgical procedures for a preexisting condition.  

 
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10.2. 
Recording of Adverse Events   
At each contact with the subject, the investigator or study staff will seek information on 
adverse events by specific questioning and, as appropriate, by examination.  
Information on all adverse events should be recorded in the source document, and also 
in the appropriate adverse event module of the case report form (CRF). All clearly 
related signs, symptoms, and abnormal diagnostic procedures results should recorded 
in the source document.  All adverse events occurring during the study period must be 
recorded.  The clinical course of each event should be followed until resolution, 
stabilization, or until it has been determined that the study treatment or participation is 
not the cause.  Serious adverse events that are still ongoing at the end of the study 
period must be followed up to determine the final outcome. Any serious adverse event 
that occurs during the year after completion of treatment will similarly be recorded and 
reported. 
10.3. 
Reporting of Serious Adverse Events 
10.3.1. 
Study Sponsor Notification by Investigator   
The study team will report serious adverse events to the DSMB, within 48 hours of first 
knowledge of the event. Serious Adverse Event (SAE) form must be completed by the 
investigator and faxed to the study sponsor within 24 hours.  The investigator will keep a 
copy of this SAE form on file at the study site. A summary report of the adverse events 
will be submitted to the IRB every six months along with the DSMB report.  
10.4. 
Termination Criteria 
The following Termination Criteria will be used to guide discontinuation of study 
medication to minimize the risks of drug-related adverse events. However, all subjects 
will be followed in conformity with intent-to-treat analytical strategy. 
 
Increase in serum creatinine >2.5 mg/dL, only if confirmed by a repeat test 
 
24-hour urinary calcium excretion rate > 400 mg; if the 24-hour collection is 
deemed erroneous by the study clinician, the test will be repeated. 
If either of these two criteria is met, the nutrition intervention will be discontinued. 
In addition, in accordance with the Endocrine Society and ISSAM guidelines for 
testosterone therapy, we will use the following termination criteria related to testosterone 
therapy:  
 
PSA increment of >1.4 ng/ml above baseline, if confirmed by a repeat test 
 
Detection of prostate or breast cancer  
 
Hematocrit >54%, if confirmed by a repeat test  
 
Myocardial infarction 

 
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 
Stroke  
If any of these criteria is met, the study medication (testosterone or placebo) will be 
discontinued.   
We recognize that other unexpected adverse events can occur. The study physician 
may stop study medication for other adverse events if in his/her opinion, continuation of 
study medication would pose an unacceptable risk to the subject’s health.  We have 
instituted a comprehensive plan for early detection of adverse events. The Data Safety 
Monitoring Board is empowered to discontinue treatment in one or more subjects, or 
halt the study, should the occurrence of adverse events so warrant. 
10.5. 
Unblinding   
Treatment assignment will be blinded to all but a single designated "unblinded" 
physician or the research pharmacist. Although testosterone treatment might increase 
the risk of certain diseases, such as prostate cancer, lower urinary tract symptoms due 
to benign prostatic hyperplasia, or erythrocytosis, the blind will not be broken even if a 
subject develops one of these conditions during the study. Instead, the following 
approach will be taken.   
a. If a subject is diagnosed with prostate cancer during the study, treatment will be 
discontinued, whether the treatment is testosterone or placebo.   
b. If the subject's score on the International Prostate Symptoms Score increases by 
≥5 points during treatment, suggesting worsening of lower urinary tract 
symptoms, the subject will be evaluated for medications that affect urine flow 
rates and for prostatitis, and treatment with an alpha blocker and urologic 
evaluation will be considered.   
c. If a subject develops a hematocrit >54%, we will recheck hematocrit. If 
confirmed, study medication will be stopped.  
11. Monitoring Subject Safety  
11.1. 
Potential Risks to Subjects   
Several conditions to which elderly men are particularly prone may be worsened by 
testosterone.  
The potential risks of the study include the risks of blood drawing, exercise testing, high 
protein intake in excess of RDA, testosterone administration, DXA scan, and the 
administration of questionnaires.  
Risks of Blood Drawing. The risks of blood drawing include pain, bleeding, and/or a 
bruise at the site of needle insertion. Serious complications such as blood clots or 
infection are very rare when proper precautions are taken.  
Potential Risks of Protein Intake in Excess of RDA.  There are no long term, 
adequately powered studies to determine the adverse health consequences of 

 
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eucaloric, high protein diets in older individuals. Several safety concerns have been 
voiced, including the risk of renal dysfunction, hypercalciurea, and osteoporosis. 
Additionally, people often perceive high protein diets as being “unhealthy” – containing 
“too much fat or animal products”.  A meta-analysis has confirmed that for every 25-g 
increase in dietary protein, urinary calcium increases by 0.8 mmol. There is concern that 
increased calcium exertion during ingestion of high protein diets may reflect bone 
demineralization and increase the risk of kidney stones. Increased amounts of dietary 
protein also might be expected to increase endogenous acid production and bone may 
be called upon to buffer the additional acid load, resulting in mobilization of bone 
calcium, and a decline in bone mineral density (BMD). A few cross-sectional studies 
have reported a positive association between dietary protein intake and hip fracture 
incidence.  However, bone balance studies have not found an increase in bone calcium 
resorption. Instead, Kerstetter et al found that additional dietary protein promotes 
intestinal calcium absorption and reduces the fraction of urinary calcium of bone origin. 
Also at odds with the bone hypothesis are a large number of epidemiological studies 
showing that a high-protein diet is associated with high, not low BMD. The net effect of 
dietary protein on bone mineral status depends on dietary availability of calcium; a diet 
rich in protein, dairy, and calcium attenuates urinary calcium and bone loss relative to a 
high carbohydrate diet.  
Additional concerns have been voiced about potential adverse effects of high protein 
diet on renal function; Brenner had hypothesized that high protein intakes by diabetic 
individuals create hyperfiltration and glomerular hypertension resulting in renal damage; 
however, this hypothesis has not found strong support. There is no evidence that high 
protein intakes up to 2 g ·kg–1 ·d–1 increase the risk of renal impairment in individuals 
with normal renal function.  However, in individuals with pre-existing impairment of renal 
function, high protein intake greater than 45% of energy intake may accelerate the 
decline in renal function. The amount of protein being investigated in this study (0.8 and 
1.3 g • kg-1 • day-1) is lower than the levels that have been associated with renal 
impairment. Also, we will exclude individuals with estimated GFR <60 ml/min.  We will 
monitor serum creatinine and 24-hour calcium excretion during the course of the study. 
Potential Adverse Effects of Testosterone.  The potential side effects of testosterone 
treatment include acne, oiliness of skin, increased growth of body hair, breast 
tenderness, a reversible increase in hemoglobin, sleep apnea, leg edema, weight gain 
and change in body habitus. Serious side effects such as hepatotoxicity and hepatic 
neoplasms, anecdotally identified with oral administration of high dose, long-term 
administration of 17-alpha-alkylated derivatives of testosterone in athletes, have not 
been observed in men when testosterone is administered parenterally or transdermally. 
In addition, there is concern and uncertainty about the long term risks of prostate and 
cardiovascular disorders in older men treated with testosterone. A recent testosterone 
trial in older men with mobility limitations reported a higher frequency of cardiovascular 
events in men assigned to the testosterone arm than in those assigned to placebo arm 
of the study (see below).   
Testosterone supplementation increases red cell mass and this is reflected in increased 
hemoglobin and hematocrit. The increment in most men with low testosterone levels is 

 
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modest, and is clinically not significant. However, in some older men, particularly in 
those who are smokers, the increase in hemoglobin might be greater. Therefore, we will 
monitor hemoglobin levels during the course of the study, and increments in hemoglobin 
levels above 54% will warrant discontinuation of testosterone treatment, referral to a 
specialist for evaluation and treatment including therapeutic phlebotomy if indicated by 
the occurrence of neuro-occlusive symptoms. In older men, testosterone administration 
has been associated with significantly greater increments in hematocrit than that 
observed in younger men. We will monitor hematocrit, and discontinue treatment, 
should hematocrit rise above 54% or should neuro-occlusive symptoms occur at any 
hematocrit. 
Testosterone administration can induce or exacerbate pre-existing sleep apnea. 
However, the frequency of this adverse event is low in young as well as older men. In 
our previous studies of carefully selected, older men, similar to those that will be 
enrolled in the proposed studies, we did not observe this complication in any participant. 
Snyder et al also did not observe a significant increase in the frequency of sleep apnea 
in older men treated with a scrotal patch for three years in comparison to those who 
received the placebo patches.    
Testosterone can also cause transient sodium and water retention; this could potentially 
cause edema or exacerbate congestive heart failure in men with pre-existing cardiac 
dysfunction. We will exclude men with CHF with Class III or IV symptoms, or those with 
myocardial infarction in the preceding 6 months. 
Although the data from experimental animals provides unequivocal evidence that 
testosterone regulates territoriality, scent marking behavior, and aggression in male 
mammals, particularly at the time of mate selection, the data in humans are highly 
equivocal. There are anecdotal reports of “roid rage” among men abusing large doses 
of androgenic steroids. However, systemic investigation has not revealed a significant 
increase in aggressive behaviors in carefully screened men given testosterone 
enanthate in doses of up to 600-mg weekly. We cannot exclude the possibility that still 
higher doses of androgenic steroids when administered to men with pre-existing 
personality disorders or psychopathology might induce aggression. In this study, we will 
use a replacement dose of testosterone gel that is expected to produce average serum 
testosterone concentrations in the mid to high normal range; we are not aware of any 
reports of “roid rage” reactions at these testosterone concentrations in either men or 
women. 
Testosterone and Prostate Cancer Risk. There is agreement that testosterone does 
not cause prostate cancer. Also, epidemiological studies have failed to demonstrate a 
consistent, dose-related, correlation between prostate cancer risk and sex hormone 
levels. However, there are a number of areas of uncertainty that are discussed below. 

 
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i. Prostate cancer is a common, androgen–dependent tumor, and androgen 
administration may promote tumor growth. Testosterone administration is absolutely 
contraindicated in men with history of prostate cancer. 
ii. Many older men have microscopic foci of cancer in their prostates. We do not know 
whether testosterone administration will make these subclinical foci of cancer grow and 
become clinically overt.  
iii. In addition, older men with prostate cancer may have low testosterone levels. 
Morgentaler et al reported a high prevalence of biopsy-detectable prostate cancer in 
men with low total or free testosterone levels despite normal PSA levels and results of 
digital rectal examination. However, this study did not have a control group, and we do 
not know whether sextant biopsies of age-matched controls with normal testosterone 
levels would yield a similarly high incidence of biopsy-detectable cancer. 
iv. Serum PSA levels are lower in testosterone–deficient men and are restored to 
normal following testosterone replacement. However, serum PSA levels do not increase 
progressively in healthy hypogonadal men with replacement doses of testosterone. In 
four placebo–controlled trials of testosterone administration in older men, the change in 
serum PSA levels over three–years was not significantly different between placebo–and 
testosterone–treated men. The increase in PSA levels during testosterone replacement 
might trigger evaluation and biopsy in some patients. 
v. More intensive PSA screening and follow-up of men receiving testosterone 
replacement might lead to increased number of prostate biopsies and detection of 
subclinical prostate cancers that would have otherwise remained undetected. 
vi. Serum PSA levels tend to fluctuate when measured repeatedly in the same 
individual over time. Therefore, when serum PSA levels in androgen deficient men on 
testosterone replacement therapy show a change from a previously measured value, 
the investigator will need to decide whether the change warrants detailed evaluation of 
the patient for prostate cancer, or whether it is simply due to test–to–test variability in 
PSA measurement.  
Testosterone Effects on the Risk of Heart Disease. The long-term consequences of 
testosterone supplementation on the risk of heart disease remain unknown. The clinical 
trials data on the effects of testosterone supplementation on cardiovascular risk are 
limited by small size of many trials, relatively short intervention periods, and poor quality 
of adverse event recording and adjudication. Meta-analyses of randomized trials have 
failed to reveal statistically significant difference between the testosterone and placebo 
arms. In the TOM Trial, a randomized, placebo-controlled testosterone trial in older men 
with mobility limitation, men assigned to the testosterone arm of the study experienced 
greater number of adverse events, particularly cardiovascular events than those 
assigned to the placebo arm of the study.  Therefore, in the proposed study, we will use 
a structured instrument for prospective recording of cardiovascular events. We will use 
a lower dose of testosterone enanthate than we had planned originally. We will exclude 
men with myocardial infarction or stroke in the preceding six months. The study duration 
is not long enough to permit meaningful evaluation of atherosclerosis progression or 
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While supraphysiological doses of non-aromatizable, 17-alpha-alkylated, orally 
administered androgens undoubtedly decrease plasma HDL-cholesterol levels, 
physiologic testosterone replacement administered either parenterally or transdermally 
has been associated with only a modest decrease in plasma HDL-cholesterol. Thus, 
previous studies in which supraphysiological doses of 17-alpha-methyl testosterone 
have been administered to women have demonstrated a decrease in plasma HDL-
cholesterol. 
Cross-sectional studies of middle-aged men find a direct, rather than an inverse, 
relationship between serum testosterone levels and plasma HDL-cholesterol 
concentrations, and an inverse correlation between testosterone levels and visceral fat 
volume in older men have demonstrated no significant change in plasma HDL-
cholesterol levels during long-term testosterone administration. Similarly, 
epidemiological studies indicate an inverse relationship between circulating androgen 
concentrations and carotid intima-media thickness; thus men and women in the highest 
quartile of androgen concentrations have the least intima media thickness. 
Spontaneous and experimentally induced androgen deficiency is associated with 
increased fat mass, and testosterone replacement decreases fat mass in older men with 
low testosterone levels. Testosterone supplementation of middle-aged men with truncal 
obesity is associated with a reduction in visceral fat volume, serum glucose 
concentration, blood pressure, and an improvement in insulin sensitivity. Surgical 
castration in rats impairs insulin sensitivity; physiologic testosterone replacement 
reverses this metabolic derangement. However, high doses of testosterone impair 
insulin sensitivity in castrated rats. These data suggest that both high and low 
concentrations of testosterone might have deleterious effects on insulin sensitivity. 
Whether variation of testosterone within the normal range is associated with risk of 
coronary artery disease remains controversial. Of the 30 cross-sectional studies 
reviewed by Alexandersen, 18 reported lower testosterone levels in men with coronary 
heart disease, 11 found similar testosterone levels in controls and men with coronary 
artery disease and 1 found higher levels of DHEAS. Prospective studies have failed to 
reveal an association of total testosterone levels and onset of coronary disease.  
Testosterone increases coronary blood flow by direct effects on blood vessel wall. In an 
intervention study, testosterone undecanoate given orally improved angina pectoris in 
men with coronary heart disease. Testosterone infusion acutely improves coronary 
blood flow in a canine model and in men with coronary artery disease. 
Testosterone levels are lower in patients with type 2 diabetes mellitus compared with 
controls. Low total and free testosterone levels are associated with a higher risk of the 
developing type 2 diabetes. Free testosterone levels are negatively correlated with 
glucose, insulin, and C-peptide levels independent of body mass index. Some of these 
studies measured free testosterone by a tracer analog method, which is not 
independent of SHBG effects. Lower testosterone levels in men are associated with 
higher levels of dense LDL particles and prothrombotic factors. 
In a mouse model of atherosclerosis that is LDL-receptor deficient surgical castration 
accelerates, and testosterone administration retards the progression of atherosclerosis. 

 
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The magnitude of testosterone effect on atherosclerosis progression is similar to that 
observed with estrogen administration. Favorable effects of testosterone on 
atherosclerosis in this mouse model are antagonized by concomitant administration of 
an aromatase inhibitor, suggesting that testosterone effects are possibly mediated 
through its conversion to estrogen in the vessel wall. Testosterone effects in retarding 
atheroslcerosis progression were independent of plasma lipids.  
Potential Risks of DXA Scanning.  The radiation exposure during a DXA scan is 
minimal (less than 25 mrems, less than that from a chest X ray). While we do not know 
whether any dose of radiation is completely safe, this amount of radiation to which the 
subjects will be exposed during the course of the study is well within the limits 
considered “safe” by federal and state regulations. 
Potential Risks of Physical Function Testing.  There is a small risk of incurring injury 
during assessment of muscle performance and physical function. This risk will be 
minimized by properly instructing the participants, and by performing these tests under 
direct supervision of an experienced exercise technician. Gordon et al have reported on 
the safety of maximal strength testing in 6,653 males and females aged 20-69 years.  
Of this sample, 1112 subjects were over 50.  Only one of the 6663 subjects experienced 
a clinically significant, non-fatal or fatal cardiovascular event in association with strength 
testing.  The authors complemented these data with two additional data sets:  1) 1819 
maximal strength assessments performed at the Cooper Clinic (Dallas, TX) and 2) 
approximately 4500 different subjects who completed approximately 20,000 maximal 
strength tests at the University of Florida’s Center for Exercise Science.  These subjects 
were aged 18-93 years. Shaw and colleagues studied maximal strength testing in 83 
elderly persons with a mean age of 66 6 years using the same 1RM methodology 
proposed for this study.  Strength in five resistance training exercises was determined in 
these subjects.   Eighty-one of the subjects completed the testing without injury, 48 
complained of muscle soreness after testing, and 2 subjects (2.4%) sustained an injury.  
Bassey using the same equipment for testing leg power proposed in this study, 
successfully tested 26 frail elderly male and female residents of a chronic care hospital,  
These subjects averaged 88.5  6 years for the men and 86.5 6 years for the women 
and had multiple pathologies.  In addition to the leg power testing, all subjects 
completed a battery of four physical function tests including a 4-step stair climb, a 6 
meter walk, and repetitive rising from a chair.  No injuries or adverse effects from the 
testing procedures was reported.  Fiatarone et al measured maximal voluntary strength 
in nonageranians without problems. Taken together, these data strongly suggest that 
maximal strength and power, and physical function testing can be performed safely in 
older men. 
Our group has extensive experience in the application of the muscle function 
assessments proposed for this study including studies in older healthy men, older men 
with COPD, and older men with low testosterone (Please, see Drs. Bhasin and Storer’s 
biosketches). We have conducted these tests in young and older men and women 
without incident for over 20 years.  We attribute this in part to the general safety of the 
tests as indicated above as well as through application of careful and systematically 
applied testing procedures.  We will conduct strength testing with Keiser pneumatic 

 
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equipment that allows as little as one pound increments in resistance, thus maintaining 
the ability to make appropriately small increments in resistance as required in older, frail 
individuals.  This equipment has been used extensively in aging research and shown to 
be safe when used with elderly persons.  The risks of strength and power testing 
include muscle soreness and injury. These risks will be minimized by following careful 
procedures (standardized protocols) for warm-ups, progressive increase in workload, 
and careful supervision of the subjects during their testing by trained and experienced 
technicians.  We will employ careful instruction, demonstration and provide practice 
sessions so that subjects may become familiar with the procedures. 
Potential Risks of Questionnaires. The instruments used for the assessment of self-
reported physical function, disability, affect, sense of well being and fatigue ask about 
personal details that the subjects might find embarrassing. Although we encourage 
subjects to answer all the questions, they have the option of not answering the 
questions that they might find embarrassing. All the questionnaires are coded and 
confidentiality is maintained at all times. 
Difficulties in Obtaining Informed Consent when there is Uncertainty about the 
Potential Risks. The purpose of the informed consent process is to provide a full 
disclosure of the potential risks so that the participants can perform a meaningful 
risk:benefits assessment. However, we also recognize that communicating this 
information effectively to research subjects is a complex and difficult job. In view of the 
growing media coverage of this issue, the research subjects may have misconceptions 
about the potential benefits. Also, the absence of definitive data makes it difficult to 
provide quantitative estimates of the risk; hence, the subjects may not comprehend the 
magnitude of the risk to their health.  
In an attempt to provide as full a disclosure as possible, we have built in a multi-step 
informational process that starts with subject’s first telephone call, and continues with 
the subject’s visit to the CTSI/GCRU when further details of the study are provided and 
the subject’s questions are answered before the subject signs the consent form. We 
recognize that the process of obtaining informed consent is a continuous process that 
does not end when the subject signs the consent form. Thus the process of providing 
information about the risks and benefits will continue throughout the course of the 
project. 
11.2. 
Procedures for Protecting Against Potential Risks 
All subjects will be monitored by frequent physical examinations, blood counts and 
chemistries. We will also monitor PSA, digital prostate examination, hemoglobin and 
hematocrit, 24-hour urinary calcium excretion, and creatinine levels. In addition, the 
Research Coordinator will be in frequent contact with the subjects. We will ascertain 
adverse events reported by the subjects at baseline, at 6 weeks, 3 months and 6 
months. 
The risks of blood drawing will be minimized in that only experienced practitioners 
(licensed physicians and clinical research nurses) will perform phlebotomy. 

 
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The risk of injury from participating in exercise testing will be minimized by several 
safety measures. The subjects will be instructed in proper technique and the risks 
minimized by careful supervision.  Patients will be screened with exercise stress testing 
to rule out cardiac ischemia and arrhythmias.  This test will be done in the presence of 
an experienced physician.  The likelihood of musculoskeletal injury from exercise testing 
will be minimized by providing a warm-up period proceeding each exercise session 
including warm-up stretching exercises.  Further, an experienced exercise trainer will 
oversee all exercise testing sessions. 
11.3. 
Data and Safety Monitoring Board 
An external DSMB will be established by the NIA to monitor all aspects of the study. 
The Board will consist of experts in geriatrics, biostatistics, clinical trials, and nutrition. 
The DSMB members will not be affiliated with the study and will be appointed by the 
NIA Director. The Board will meet every six months to review subjects’ safety, study 
progress and data integrity and completeness. After each meeting, the DSMB will 
provide the NIA Director with its recommendations, and the Director will decide whether 
to accept them. The study team will submit the DSMB report to the Partners IRB.   
12. Data Management 
The Boston University Data Coordinating Center will provide data management and 
statistical programming under the supervision of the investigators and biostatistician.  
This will be accomplished by the successful completion of the following tasks: 
Design Randomization through a Web Application:  An automated randomization 
application will be available.  Upon completion of Inclusion/Exclusion form, a new 
subject ID will be generated and the randomization table, stratified by diabetes status 
will be queried for study arm designation. 
Design database for subject enrollment and tracking: A database system will be 
designed to track subjects, provide documentation of eligibility and agreement to 
participate or the reason for refusal, and, whether the subject signed the informed 
consent document. The database will provide automatic alerts to the study coordinators 
about due dates for follow-up visits. The database will reside on a secure, password 
protected SQL server; only connections from users authenticated from the domain 
controller will be accepted, providing a secure environment. The database will be 
automatically backed up on a nightly basis.   
Design data collection forms:  The Data Management team will prepare data 
collection forms creating a standardized data collection infrastructure.  Forms will be 
designed to ensure that all data fields are unambiguous, items are easily read, 
response categories are in standardized units, and the instructions are clearly worded.  
Data Capture and Quality Control:  The completed forms will be entered into a web-
based database system. A unique ID will be used to identify individual forms; subject 
names will not be recorded. After verification, the data will be saved to the database 
(Microsoft SQL server). If an error is detected, the form will not be committed to the 

 
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database until resolved. After forms have been verified, computer algorithms will be 
written in SAS to check internal inconsistencies.  
Dietary data will be entered by a research nutritionist into the Nutrition Data System 
(NDS) of the University of Minnesota. Similarly, the 24-hour food recalls collected during 
the study will be reviewed and the subject debriefed prior to entry of the data by a 
research nutritionist into the Nutrition Data System (NDS) of the University of 
Minnesota. 
Create analytic datasets: All dietary data will be cleaned by an experienced nutrition 
data manager. A statistical programmer will be responsible for cleaning other data, as 
well as creating analytic data sets and carrying out data analyses. Documentation will 
include the creation of standardized protocols for coding and entering dietary and other 
data, creation of a data dictionary that includes all original and composite or created 
variables. The statistical programmer, under the supervision of the project Statistician, 
will write the SAS code for all analyses for DSMB reports, progress reports, and 
manuscripts. 
Data Security: The Data Management Team has in place standard procedures for 
database backup to prevent data loss, systems validation, and performance monitoring. 
User access will be controlled by assignment of usernames and passwords. The DCC 
systems meet the regulatory requirements for electronic records described in Code of 
Federal Regulations (CFR) 21 Part 11, and the applicable regulatory guidelines.  
Maintaining Blinding. The Data Management Team will collaborate with the 
Investigational Drug Service and the unblinded physician to protect the blinding of 
treatment assignments. Electronic access to information that could indirectly or directly 
lead to unblinding will be limited and such information will be stored in password-
protected files. 
12.1.1. 
Maintaining Anonymity of Submitted Medical Records   
Clinical site personnel will de-identify all medical records before sending them to the 
DCC by obliterating any Protected Health Information (PHI). Upon receipt, DCC 
personnel will review the records to ensure that no PHI is visible. 
12.1.2. 
Confidentiality 
Information about study subjects will be kept confidential and managed according to the 
requirements of the Health Insurance Portability and Accountability Act (HIPAA) of 1996.   
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