Long-term Effects of Weight Loss and Supplemental Protein on Physical Function
Phase: NA
Conditions: Obesity, Weight Loss, Diet Modification, Aging, Weight Change, Body, Dietary Habits
Sponsor: Wake Forest University Health Sciences

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Title: Long-term Effects of Weight Loss and Supplemental Protein on Physical Function 
NCT03074643 
Date: 08/02/2022  
 

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Scientific Study Title:  Long-term effects of weight loss and supplemental protein on physical function 
Lay Title: UPLIFT (Utilizing Protein During Weight Loss to Impact Physical Function) 
Principal Investigator:  Denise Houston, PhD 
Co-investigator(s):  Tina Brinkley, PhD, Steve Kritchevsky, PhD, Iris Leng, PhD, Mary Lyles, MD, Barbara 
Nicklas, PhD 
Sponsor or funding source:  NIH/NIA 
Background, Rationale and Context 
Obesity exacerbates age-related declines in physical function and is a strong determinant of mobility 
disability.1-3 One-third of older adults are obese;4 thus, there is an urgent need to identify effective therapies 
that prevent obesity-related declines in physical function. The loss of lean mass with age contributes to age-
related declines in physical function.5-7 Since approximately one-fourth of lost weight is comprised of lean 
mass,8-11 current weight loss recommendations regarding macronutrient composition ‒ and protein in particular 
‒ during caloric restriction may exacerbate the loss of lean mass in older adults, further compromising physical 
function over the long-term. Moreover, studies suggest that older adults need more protein to elicit the same 
stimulatory effect on muscle protein synthesis and maintain lean mass than younger adults.12-16 Thus, 
recommendations for effective weight loss treatments that preserve lean mass and optimize function in obese 
older adults may differ from those for younger adults. Additionally, recent Guidelines (2013) for the 
Management of Overweight and Obesity in Adults stated “there is a need for further research to understand the 
most appropriate strategies and prescriptions for weight loss [in] older adults.”17  
Limited evidence in older adults shows that weight loss improves physical function while in the weight-reduced 
state.8;18-24  However, most people are not successful at long-term maintenance of weight loss.25 Whether 
improvements in physical function persist is unknown, particularly if weight regain occurs. We and others have 
shown that weight regain after weight loss resulted in greater regain of fat relative to lean mass a year after the 
intervention.26;27 Consequently, weight loss and subsequent regain may lead to even less relative lean mass 
and worse physical function. This problem highlights the need to identify a strategy that preserves lean mass 
during weight loss and promotes the deposition of lean mass following weight loss. Similar to studies in 
younger adults,28 we previously found that a higher protein diet during caloric restriction may be a key 
determinant for retaining lean mass during weight loss in postmenopausal women.10;29 A higher protein diet 
after weight loss may also enhance weight loss maintenance by reducing overall weight regain, in part, by 
favoring regain of lean mass over fat.30 Thus, the potential benefits of a higher protein diet during and 
subsequent to weight loss in obese older adults should translate into even greater improvements in physical 
function over both the short- and long-term; however, this needs to be definitively tested.   
Objectives 
Our primary goal is to determine the effect of a higher protein (1.2 g/kg body wt/d; ~30% of energy) / lower 
carbohydrate (CHO; 40-45% of energy) diet during a 6-month weight loss intervention in obese older adults on 
physical function compared with an isocaloric lower protein (current RDA of 0.8 g/kg body wt/d; 15-20% of 
energy) / higher CHO (55-60% of energy) diet, and whether continuing a higher protein / lower CHO diet 
following weight loss results in better maintenance of physical function. This will be accomplished with an 18-
month randomized trial in 225 obese (BMI 30-45 kg/m2 or 27-30 kg/m2 with 1 additional risk factor), older (65-
85 years) men and women at risk for disability (expSPPB <2.50). All participants will undergo a 6-month weight 
loss intervention of caloric restriction and exercise followed by 12 months of follow-up with randomization to 
one of three groups (n=75/group): 1) Lower protein / higher CHO diet for the 6-month weight loss and 12-
month follow-up phases (RecProt); 2) Higher protein / lower CHO diet for the 6-month weight loss phase only 
(6-mo HiProt); or 3) Higher protein / lower CHO diet for the 6-month weight loss and 12-month follow-up 
phases (18-mo HiProt).  
Primary Aim: To determine the effects of a higher protein / lower CHO diet vs. lower protein / higher CHO diet 
during a 6-month weight loss intervention and 12 months of follow-up on 18-month physical function assessed 
by the expanded Short Physical Performance Battery (primary outcome) and muscle strength. 

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Hypothesis: Participants randomized to a higher protein diet during weight loss and follow-up (18-mo 
HiProt) will have greater improvements in physical function than participants randomized to a higher protein 
diet during weight loss only (6-mo HiProt), followed by those randomized to a lower protein diet (RecProt). 
Secondary Aim: To determine the effects of a higher protein / lower CHO diet vs. lower protein / higher CHO 
diet during a 6-month weight loss intervention and 12 months of follow-up on 18-month body weight and body 
composition. 
Hypothesis: Participants randomized to a higher protein diet during weight loss and follow-up (18-mo 
HiProt) will have better weight loss maintenance, lower fat-to-lean mass ratio, greater thigh muscle volume, 
and lower inter-muscular adipose tissue than participants randomized to a higher protein diet during weight 
loss only (6-mo HiProt), followed by those randomized to a lower protein diet (RecProt). 
We will also assess the effects of a higher protein diet on: 1) 6-month changes in physical function, weight, and 
body composition; 2) 6- and 18-month changes in bone mineral density; and 3) 6- and 18-month changes in 
cardiometabolic risk factors (e.g., glucose and lipids); and 4) whether the effects of a higher protein diet on 
lean mass mediates the effects on physical function at 6- and 18-months.  
Methods and Measures 
Study Overview: 
We will use a 3-group design in 225 obese (BMI 27-30kg/m2 with 1 risk factor or BMI 30-45 kg/m2), older (65-
79 years) men and women at risk for disability (expSPPB <2.50) who will undergo a 6-month weight loss 
intervention followed by a 12-month follow-up phase to test our overall hypothesis that a higher protein (1.2 
g/kg body wt/d) / lower CHO diet during a 6-month weight loss intervention improves physical function 
compared with an isocaloric lower protein (the current RDA, 0.8 g/kg body wt/d) / higher CHO diet and 
continuing a higher protein / lower CHO diet for 12-months following weight loss will result in better 
maintenance of improved physical function. All participants will undergo a 6-month weight loss intervention 
involving caloric restriction and supervised exercise followed by 12 months of follow-up with randomization to 
one of three groups (n=75/group):  1) Lower protein / higher CHO diet for the 6-month weight loss and 12-
month follow-up phases (RecProt); 2) Higher protein / lower CHO diet for the 6-month weight loss phase only 
(6-mo HiProt); or 3) Higher protein / lower CHO diet for the 6-month weight loss and 12-month follow-up 
phases (18-mo HiProt). Physical function (primary aim) will be assessed by the expanded Short Physical 
Performance Battery (expSPPB) and muscle strength and body composition (secondary aim) assessed by 
dual-energy X-ray absorptiometry (DXA) and computed tomography (CT) at baseline, 6- and 18-months. At the 
same time points, we will also measure bone mineral density (DXA) and cardiometabolic risk factors.  
Recruitment of study participants: 
We plan to recruit, screen, and enroll 225 participants in Years 1-3 (~1-2 per week). Our recruitment goals 
include: 30% male and 25% minority. We will target men and women ≥65 years who live in the Piedmont Triad, 
including the Greensboro metropolitan area (a 20-30 min drive from the Medical Center). We will use 
community-based recruitment strategies developed by the Clinical Research Core of the WFU Pepper Center, 
including direct mail, newspaper advertisements, and cross-study referrals from other on-going studies. 
Specific inclusion/exclusion criteria (see Table 1) are in place to eliminate those that may be adversely 
affected by the interventions or that are not able to comply with the interventions. To reduce the 
potential adverse effects of WL, we will exclude individuals that are non-obese, those with recent WL, those 
who have osteoporosis, and/or anyone on medications that may affect bone density. We also exclude anyone 
who cannot walk unassisted. 
Participant screening and randomization: 
All individuals who respond to our recruitment strategies will call a toll-free phone number and a recruiter will 
describe the study and perform a brief screen for general eligibility (see Table 1). They will be asked about 
their age, weight, height, current physical activity habits, body weight in the past year, smoking history, medical 
history, and current medications. All participants must conform to the inclusion/exclusion criteria.  
Individuals who pass the telephone screening will be scheduled for an in-clinic screening visit (SV1) which is 
conducted in the early morning following an overnight (8 hr) fast. Before any data collection, all participants will 
provide written informed consent and complete a HIPAA authorization form in accordance with the WFSM IRB 
policies. At this visit, weight and height will be measured to calculate BMI. After confirmation of meeting the 

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BMI inclusion criteria, individuals will undergo further screening including the expanded Short Physical 
Performance Battery (expSPPB), a cognitive screen (Montreal Cognitive Assessment, MoCA31), physical 
activity assessment (the Community Healthy Activities Model Program for Seniors [CHAMPS] questionnaire32) 
to ensure no regular participation in resistance exercise or high-volume aerobic training in the past 6 months, a 
depressive symptom screening test (Center for Epidemiological Studies-Depression, CES-D33), a weight loss 
readiness questionnaire, and, if still eligible, a medical history, a comprehensive review of medications, blood 
pressure, and a fasting blood draw for a metabolic screening panel and complete blood count (CBC) to 
exclude those with recent history of coronary heart disease, cancer, liver or renal disease, severe pulmonary 
disease, gross physical impairment, uncontrolled hypertension, uncontrolled diabetes, or any contraindications 
to exercise. Participants will also be given a 4-day food record to complete. If still eligible, a second in-clinic 
screening visit will be scheduled for a GXT (to rule out any contraindications for supervised exercise) and DXA 
scan (to rule out osteoporosis) and to collect the 4-day food record. Participants will also do a taste test of the 
protein and carbohydrate supplements to ensure that they are willing to consume the product during the 
intervention and complete a perceived weight loss readiness questionnaire.  
Table 1. Inclusion/exclusion Criteria 
Criteria 
Inclusion 
Exclusion
Assessment
Age 
65-85 years  
 
Self-report 
Obesity status 
BMI: 30-45 kg/m2 or 
BMI 27.0 – <30.0 AND at 
least ONE of the following 
risk factors:  
1)      elevated waist 
circumference (>35 inches in 
women, >40 inches in men)  
2)      diabetes,  
3)      hypertension,  
4)      dyslipidemia,  
5)      or other obesity-related 
comorbidities: clinically 
manifest coronary artery 
disease [e.g., history of MI, 
angina pectoris, coronary 
artery surgery, coronary 
artery procedures (e.g., 
angioplasty) if not within the 
past year], other 
atherosclerotic disease [e.g., 
peripheral arterial disease, 
abdominal aortic aneurysm, 
symptomatic carotid artery 
disease if not within the past 
year], sleep apnea, or 
osteoarthritis of the knee or 
hip. 
 
Measured on scale 
 
 
1)Measured with tape 
measure 
 
2-4) Self-report and 
treatment 
 
5) Self-report 
Weight loss 
 
Weight loss (≥5%) in past 6 months 
Self-report 
Physical activity status No regular (moderate intensity 
or greater) resistance training 
and/or aerobic exercise (>20 
mins/d) for past 6 months  
 
Self-report 
Functional status 
expSPPB <2.50 
Dependent on cane or walker 
expSPPB / self-report 
Cognitive status   
 
Cognitive impairment (MoCA score <22) 
Questionnaire 
Orthopedic status  
No contraindications for safe 
and optimal participation in 
exercise training 
Severe arthritis, or other musculoskeletal 
disorder 
Joint replacement or other orthopedic 
surgery in past 6 months 
Self-report  
Co-morbidity/health 
status 
Approved for participation by 
Medical Director (Dr. Lyles) 
Uncontrolled resting hypertension (>160/90 
mmHg); 
BP measurement 
 
 
Uncontrolled diabetes (HbA1c ≥8.0%) 
HbA1c blood test 
 
 
Current or recent past (within 1 year) 
severe symptomatic heart disease, 
Self-report  

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Table 1. Inclusion/exclusion Criteria 
Criteria 
Inclusion 
Exclusion
Assessment
uncontrolled angina, stroke, chronic 
respiratory disease requiring oxygen; 
uncontrolled endocrine/metabolic disease; 
neurological or hematological disease; 
cancer requiring treatment in past year, 
except non-melanoma skin cancers; liver or 
renal disease; or clinically evident edema  
 
 
Unstable Severe Depression 
Questionnaire (CES-D) 
 
 
Serious conduction disorder, uncontrolled 
arrhythmia, or new Q waves or ST-segment 
depressions (>3 mm at rest or ≥2 mm with 
exercise) 
GXT 
 
 
Abnormal kidney function (eGFR <30 
based on serum creatinine, age, gender, 
and race) 
Metabolic panel 
 
 
Anemia (Hb<13 g/dL in men; <12 g/dL in 
women) 
CBC 
 
 
Drug abuse or excessive alcohol use (>7 
drinks/week women; >14 drinks/week men) 
Self-report 
 
 
Use of any tobacco or nicotine products in 
the past year 
Self-report 
 
 
Osteoporosis (T-score < -2.5 on hip or 
spine scan) 
DXA 
Medication / 
supplement use  
 
Regular use of growth hormone, anabolic 
steroids/hormones, sex steroids 
(testosterone; estrogen – oral or patch) or 
corticosteroids (medications that can affect 
muscle mass/strength and/or bone)  
Osteoporosis medication  
Protein supplements (and unwilling to stop 
using for duration of study) 
Weight loss medications or procedures 
Self-report and 
medication inventory 
Research participation Willing to provide informed 
consent 
Current participation in another intervention 
study 
Self-report 
 
Agree to all study procedures 
and assessments 
 
 
 
Willing to consume 
protein/CHO supplements for 
up to 18 months 
 
Taste test 
 
Able to provide own 
transportation to study visits 
and intervention sessions 
 
 
 
 
Individuals who meet all eligibility criteria and provide informed consent will be randomized to one of the three 
intervention groups (following all baseline testing) using a web-based randomization scheme (developed by Dr. 
Leng) with blocking stratified by sex. Block size and the randomization sequences will be unknown to the 
research staff. Treatment assignment will be generated using a program that the study coordinator will access 
from her PC via the study website. Individuals with abnormal results at screening will be referred to their 
physicians. 
Study Interventions 
6-month weight loss intervention: 
Caloric restriction and behavior modification/nutrition education:  All participants will undergo a dietary weight 
loss intervention designed to elicit behavioral changes leading to decreased caloric intake sufficient to yield a 
~10% loss of initial body mass. Recommended weight loss interventions for obese older adults include 
moderate caloric restriction (CR) to elicit a weight loss of 0.5-1 kg/wk.1;34-36 Thus, the 6-month weight loss 

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intervention is designed to elicit ~0.5 kg/wk weight loss through a CR level of -500 kcal/d, plus an exercise 
prescription that meets current American Heart Association (AHA) and American College of Sports Medicine 
(ACSM) physical activity recommendations for older adults (described below).37 With a calorie deficit of 500 
kcal/d, we expect total weight loss of ~10.0 kg. The reduced calorie level is derived by subtracting 500 from 
each persons estimated daily energy needs for weight maintenance. Individual energy needs are calculated 
from the direct measurement of resting metabolic rate and applying an activity factor based on a description of 
their daily activities (typically 1.3 for sedentary persons). No woman will be recommended <1100 kcals/d and 
no man <1300 kcals/d.  
The weight loss intervention will incorporate nutrition education (via group and individual meetings with the 
study dietitian), self-monitoring skills, cognitive-behavioral strategies for promoting lifestyle behavior 
modifications, and planned meals. Behavioral and educational group and individual sessions will be held 
weekly in a 3:1 ratio (3 group and 1 individual session per month). Group sessions will be held in-person in 
small groups (maximum of 6 participants per group) to maintain physical distancing (with the option to go 
entirely remotely via WebEx or phone if the conditions arise – e.g., increase in COVID-19 community spread, 
new restrictions on in-person research are put in place, and/or if stay at home orders are put back in place). 
Individual sessions will be held in-person with the option of remote sessions (via WebEx or phone) if the 
conditions arise. The diet intervention will be based on participants choosing and preparing their own meals 
from the meal plans. Sessions will be led by the study dietitian and/or interventionist with expertise in 
behavior therapy with older adults. All participants will be instructed in behavior modification with advice on 
food selection, portion size, relapse prevention, and self-monitoring techniques. Emphasis will be placed on 
following the menus provided. The behavioral sessions will focus on awareness of changing eating habits to 
lower caloric intake. Factual information regarding what food changes to make, how to make them, and why 
they are important will be explained. Each group session will include problem solving. During the individual 
sessions, the study dietitian will review individual progress, solve problems, answer questions, and set goals. 
Examples of topics to be covered include calorie balance, emotional eating, eating out, self-monitoring, 
reacting to environmental and social cues, portion control, and relapse prevention. All participants will be 
taught self-monitoring techniques for diet and body weight. They will be asked to track their daily intake of all 
foods and beverages in a log book provided to them, and given feedback on their log books at each session.   
Meal plans will be developed based on estimated calorie levels in 100 calorie increments (1100 calories, 
1200 calories, 1300 calories, etc) to attain the weight loss goal. Each meal plan will have up to 7 
interchangeable choices at each meal (breakfast, lunch, dinner) and snack (midday and evening). At each 
meal, participants within a given calorie level will select foods from the list to prepare and consume. There will 
also be choices that allow for eating meals at restaurants that meet the specified guidelines. Meals will differ 
between calorie levels in portion size and types of foods. In addition, there will be different selections for 
those on the higher protein / lower CHO diet vs. the lower protein / higher CHO diet (see below). The food 
plan will allow for individual preferences for various food items, and the study dietitian will work with 
participants to tailor the diet to individual needs. Participants will be advised to take calcium (500-1000 mg/d) 
and vitamin D (800 IU/d) supplements to ensure adequate intake of these nutrients.   
Body weight will be monitored and recorded weekly. Participant logs of everything they eat or drink will be 
reviewed weekly by the study dietitian to verify compliance to the diet. If a participant is not meeting weight 
loss goals (~0.5 kg/wk), calorie intakes will be individually modified to produce the desired rate of weight loss.  
If it is evident, either through diet logs or inadequate weight loss (~0.5 kg/wk), that participants are not 
compliant, individual counseling sessions with the study dietitian will be held to improve compliance. For 
participants who have difficulty in achieving the weight-loss and/or protein intake goals, we will use a 
behavioral “toolbox” that includes frequent individual behavioral counseling sessions, prescription of meal 
replacements, incentives, home-assessments, or other items deemed appropriate. Diet plans may be altered 
to restrict snacks and/or the size of meals to meet weight loss goals.   
Macronutrient distribution:  Those in the higher protein / lower CHO diet groups (6-mo HiProt and 18-mo 
HiProt) will be prescribed a diet containing more protein than those in the lower protein (the current RDA) / 
higher CHO diet group (RecProt). To ensure the diets are isocaloric, this protein will replace part of the 
carbohydrate prescribed in the lower protein / higher CHO diet group. Individuals assigned to the lower protein 
/ higher CHO diet will have a targeted protein intake of 0.8 g/kg body wt/d (~15-20% of energy), 25-30% of 
energy from fat (<10% from saturated fat), and 55-60% of energy from carbohydrates. The higher protein / 
lower CHO diet groups will have a targeted protein intake of 1.2 g/kg body wt/d (~30% of energy) with a 

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reduction in CHO to 40-45% of energy. Both the higher and lower protein diet groups fall within the Dietary 
Reference Intakes for macronutrients including protein (10-35% of energy).38 Fat intake will remain similar 
between groups while CHO will fluctuate to compensate for differences in energy from protein between groups.  
Participants in the higher protein / lower CHO diet groups will consume a protein supplement daily (NutraBio™ 
100% Whey Protein Isolate) which will be provided throughout the study. The supplement will provide ~50 g of 
high quality protein containing 22.6 g of essential amino acids, 10.7 g branch chain amino acids consisting of 
5.2 g of leucine, 3.0 g of isoleucine, 2.5 g of valine, and 200 calories.  Since protein intake tends to be skewed 
towards higher amounts at the evening meal39 and data suggest a protein threshold of 25-30 g protein 
containing ~2.5-2.8 g of leucine/meal optimizes protein synthesis,40-42 participants will be instructed to consume 
25 g (1 scoop) of the protein supplement twice daily with their morning and midday meal/afternoon snack. 
Participants in the lower protein / higher CHO diet group will consume a carbohydrate supplement daily 
(NutraBio CarboMax; provided throughout the study) which will provide ~50 g of maltodextrin and 200 calories 
to maintain blinding during the 6 month weight loss intervention. 
At the first individual meeting with the study dietitian following randomization, the study dietitian will provide 
each participant with a 6-month supply of protein or carbohydrate supplement and provide oral and written 
instructions for using the study supplement. The study dietitian will also provide a 6-mo supply of protein for 
those in the 18-mo HiProt group at the end of the 6 month weight loss intervention and at the 6-mo post-weight 
loss follow-up visit. 
Exercise Intervention:  All participants will be expected to participate in a supervised, center-based exercise 
program involving moderate-intensity aerobic exercise (e.g., treadmill walking) 3 days/wk during the first 6 
weeks of the 6-month weight loss intervention in accordance with the AHA and ACSM physical activity 
recommendations for older adults.37 All participant supervised, center-based exercise sessions will be 
scheduled in advance to ensure that physical distancing can be maintained (maximum of 5 participants per 
session). Supervised exercise sessions will occur in our research exercise facility under the direction of an 
exercise interventionist to minimize individual variability in compliance and progression. Injuries will be 
minimized by a feasible progression of exercise duration and careful monitoring of proper footwear and 
stretching exercises. Each exercise session will begin and end with 3-5 min of walking at slow pace and light 
stretching. The aerobic training program will progress from 15-20 min at 50% heart rate reserve (HRR; based 
on age-predicted maximal heart rate) the 1st week to 40 min at 65-70% HRR by the end of the 6th week. Heart 
rate (HR) will be measured before each exercise session and at least two HR readings (measured by Polar 
heart rate monitors) will be taken during aerobic exercise. Treadmill speed and grade will be adjusted as 
necessary to ensure that participants exercise at their prescribed exercise intensity. Treadmill speed and 
grade, heart rate, exercise duration, and amount of energy expended will be recorded for each exercise 
session. After the first 6 weeks of the 6-month weight loss intervention, participants will continue center-based 
exercise 1-2 days/wk and exercise on their own 1-2 days/wk for a total of 3 days/wk combined center- and 
home-based exercise (with the option of transitioning to all home-based exercise if the conditions arise – e.g., 
increase in COVID-19 community-spread, new restrictions on in-person research are put in place, and/or if 
stay at home orders are put back in place). Participants will be taught how to rate their perceived exertion so 
that their home-based exercise mirrors that done during their supervised, center-based exercise sessions 
(target RPE between 13 and 15) and given tips on how to exercise safely on their own. Participants will be 
asked to keep exercise logs detailing the date, type, duration and their rating of perceived exertion for each 
home-based exercise session. Compliance to the exercise prescription (attendance/ frequency, intensity and 
duration) will be monitored. The goal for attendance at center- and home-based exercise sessions is >80% 
and participants who miss >3 sessions per month will be counseled to improve exercise attendance.    
12-month follow-up:  At the end of the 6-month weight loss intervention, the study dietitian will meet individually 
with all participants to devise a weight maintenance strategy, including sample meal plans. Participants will be 
encouraged to use relapse-prevention techniques, e.g., managing the environment, adjusting appropriate goal-
based daily energy needs, and continuing self-regulatory skills. At the 6-month post-weight loss intervention 
follow-up visit, participants will again meet individually with the study dietitian, review their weight maintenance 
goals and strategies, and make modifications as needed. Participants randomized to a higher protein / lower 
CHO diet during the 12-month follow-up period (18-mo HiProt) will be provided with a protein supplement (see 
above) to consume daily and counseled on how to include the protein supplement in their maintenance meal 
plan. Participants who have difficulty consuming the protein supplement will be given the option to reduce the 

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frequency of the protein supplement by increasing dietary protein from food sources. Group seminars on 
health-related topics will be held quarterly to keep participants engaged in the study. 
Schedule and organization of assessment visits 
All assessments will be conducted during 7 visits (two screening, two baseline, one 6-month post-weight loss 
intervention, one 12-month follow-up, and one 18-month follow-up visits; Table 2). The nature, purpose, and 
risks of all tests will be explained to participants prior to obtaining their written consent and prior to each test.  
 
Table 2.  Assessment              Visit    
timeline                                    Code   
SV1 
SV2 
BV1 
BV2 
 
FV1/2 
 
FV3 
(12-mo) 
 
FV4/5 
 
 
 
 
 
 
(6-mo) 
 
 
(18-mo) 
Informed consent/HIPAA form,  
weight, height, waist circumference, 
blood pressure, expanded SPPB, 
demographics, cognitive screen 
(MoCA), depression screen (CES-
D), weight loss readiness 
questionnaire, fasting screening 
blood draw (CMP and CBC),  
CHAMPS physical activity 
questionnaire, medical history and 
medication review, instructions on 
how to keep a 4d food record 
X 
 
 
 
6-month intervention 
 
6-month follow-up 
 
12-month follow-up 
 
Collection of 4d food record 
 
X 
 
 
 
 
 
Review Inclusion/Exclusion criteria 
X 
X 
 
 
 
 
 
Protein/CHO taste test 
 
X 
 
 
 
 
 
Medication review 
 
X 
X 
X 
X 
X 
X 
Weight  
 
X 
X 
X 
X 
X 
X 
Blood pressure 
 
 
 
X 
X 
X 
X 
RMR  
 
 
 
X 
X 
 
(X)*** 
Arterial stiffness 
 
 
 
X 
X 
 
(X)*** 
GXT and Perceived Fatigue 
 
X 
 
 
(X)** 
 
 
Expanded SPPB*, leg strength, grip 
strength, and 400-m walk  
 
 
X 
 
X 
X 
X 
24-hr dietary recalls  
 
 
X 
X 
X 
 
X 
Fasting blood draw 
 
 
 
X 
X 
 
X 
Anthropometrics 
 
X 
 
 
X 
 
X 
DXA scans 
 
X 
 
 
X 
 
X 
CT scans 
 
 
X 
 
X 
 
X 
CHAMPS physical activity 
questionnaire 
 
 
 
 
X 
X 
X 
MAT-sf 
 
 
X 
 
X 
X 
X 
CES-D 
 
 
 
 
X 
X 
X 
SF-36 
 
 
X 
 
X 
X 
X 
DSST 
 
 
X 
 
X 
 
X 
Fatigue questionnaire (FACIT) 
 
 
X 
 
X 
X 
X 
Instructions for 24-hr urine sample 
collection†  
 
 
X 
 
X 
 
X 
Return 24-hr urine sample† 
 
 
 
X 
X 
 
X 
* Expanded SPPB at FV1/2, FV3, and FV4/5 only. 
† Collected from ppts after FV1/2; delivered to ppts prior to FV4/5. 
** Dropped after COVID19 in-person research pause to allow us to combine FV1 & FV2 visits 
*** Dropped after COVID19 in-person research pause to allow us to combine FV4 & FV5 visits 

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All assessments will take place by study staff blinded to the participant’s treatment assignment. To ensure that 
staff remain blinded, participants will be asked not to discuss their intervention with the assessor. Additionally, 
the staff member will remind the participants not to tell them which intervention group they have been assigned 
to.   
The first screening visit (SV1) and the resting metabolic rate (RMR; BV2 and FV1/2 (6-mo)) and fasting blood 
draw visits (BV2, FV1/2 (6-mo), and FV4/5 (18-mo)) will occur in the early morning after an 8 hour fast. After a 
blood draw for the metabolic screening panel and CBC (SV1), RMR (BV2 and FV1/2), and fasting blood (BV2, 
FV1/2, and FV4/5), participants will be given a light breakfast. BV1 and BV2 will occur approximately one week 
apart. The follow-up visits will occur (1) after the 6-month intervention (FV1/2; weeks 27-32), (2) 6 months post-
intervention (FV3; weeks 53-56), and (3) 12 months post-intervention (FV4/5; weeks 79-84).  
The first screening visit (SV1) will be conducted in the morning following an 8 hour overnight fast for 
measurement of a complete metabolic panel and CBC for screening purposes. After blood pressure, pulse, 
height and weight are collected and the blood draw completed, participants will be given a snack and continue 
with the remainder of the visit. First, the expanded Short Physical Performance Battery (expSPPB)43 will be 
administered and scored. If still eligible, participants will then undergo a cognitive screen (assessed using the 
Montreal Cognitive Assessment, MoCA16), a depression screen (assessed using the Center for 
Epidemiological Studies-Depression (CES-D)), a weight loss readiness questionnaire, and a medical history 
and review of medications and dietary supplements. The CHAMPS Physical Activity Questionnaire for Older 
Adults32 will be administered to estimate physical activity in a typical week over the past 4 weeks. 
Demographics will also be collected. Potentially eligible participants will be given instructions on how to keep a 
food diary, and asked to record four days of food intake prior to the second screening visit. Following the first 
screening visit, the study coordinator will check the lab results to determine if the participant is still eligible 
based on blood work. If the participant is eligible, they will be notified and scheduled for a second screening 
visit. If the participant is not eligible, they will be notified and given a copy of their screening blood work.  
At the second screening visit (SV2), participants will undergo an assessment of Perceived Fatigue on the 
treadmill as a warm-up to acclimate participants to the treadmill. Then participants will do a graded exercise 
stress test (VO2 max) using a Ramp protocol44 to exclude those with exercise-induced ischemia. All 
participants must be free of any contraindications for participation in an exercise program according to the 
American College of Sports Medicine criteria.44 These criteria will eliminate those with conditions that may 
affect their ability to complete a 6-month exercise program. Whole-body, AP spine, and hip DXA scans will be 
conducted to exclude persons with osteoporosis. Any individual with abnormal test results at their screening 
visit will be referred to their physician. The participant will have anthropometric measurements taken at their 
waist, hip and thigh. Food records will be collected and evaluated to insure participants will be willing to record 
dietary intake during the 6-mo weight loss phase and provide baseline dietary preferences to the study RD. 
Participants will be given a taste test of the protein and carbohydrate supplements to ensure they are willing to 
consume them for the duration of the study. Participants who are eligible to participate in the study will be 
randomized to one of the 3 study intervention groups (following all baseline testing) using a web-based 
randomization scheme (from Dr. Leng) with blocking stratified by gender. 
At the first baseline visit 1 (BV1), weight, lower extremity muscle strength (Biodex dynamometer), grip 
strength, and 400-m walk time will be measured. Any changes to medical history and current medications and 
dietary supplements will be reviewed. Then the Mobility Assessment Tool for Disability – short form (MAT-sf) to 
assess difficulty performing daily tasks, the Short Form 36 (SF-36) to assess quality of life, the Digit Symbol 
Substitution Test (DSST) to assess attention and psychomotor speed, and the FACIT questionnaire to assess 
fatigue will be administered. The participant will also have a series of CT scans (abdomen, hip and thigh) at 
this visit. Participants will be given a 24-hr urine collection bottle and instructions for collecting urine. 
Participants will be scheduled for their BV2 visit approximately one week later.  
Baseline visit 2 (BV2) will take place approximately 1 week after the BV1 visit in the morning (after an 8 hour 
overnight fast). Participants will have their weight and blood pressure measured. Then participants will have 
their resting metabolic rate (RMR) assessed for the purpose of estimating each person’s baseline calorie level. 
Participants will also undergo tests to assess arterial stiffness, including aortic augmentation index (AIx) and 
arterial wave reflection magnitude. Participants will then have blood drawn for glucose, insulin, HbA1c, lipids, 
and storage. The first of three 24-hr dietary recalls will be administered to introduce and train the participant for 
the procedures used in subsequent 24-hr dietary recalls done by phone (in the event that scheduling precludes 

V08.02.22 
 
 
 
10 
administration of the first 24-hr dietary recall in person, the participant will be given written instructions and the 
first 24-hr dietary recall administered by phone). Two additional 24-hr diet recalls will be administered by phone 
following BV2 but prior to the participant’s first intervention visit.  (24-hr diet recalls may be started at BV1 for 
some participants to ensure they are completed prior to the start of intervention.)  Following all fasting 
measures, participants will be given a snack. Medical history and current medications and dietary supplements 
will be reviewed. The 24-hour urine sample will be collected to measure urinary nitrogen and creatinine for 
estimating dietary protein intake and for storage.  
Randomization/first intervention visit. Due to the group nature of the diet classes, participants will be 
randomized in waves with all participants starting intervention in a given week. The study interventionist will 
check to see which intervention group the participant is randomized to at their first meeting with each eligible 
participant during the first week of each wave’s intervention.  
The 6-month follow-up visit (FV1/2) will take place following week 26 of the intervention in the morning after 
an 8 hour overnight fast. Participants will have their weight and blood pressure measured. Then participants 
will have their resting metabolic rate (RMR) measured for the purpose of estimating each person’s weight 
maintenance calorie level. The participant will also undergo tests to assess arterial stiffness. Blood will be 
drawn for glucose, insulin, HbA1c, lipids, CMP and CBC, and storage. Participants will then be provided with a 
snack. Any changes to medical history and current medications and dietary supplements will be reviewed. 
Whole-body, AP spine, and hip DXA scans will be done. The participant will have anthropometric 
measurements taken at their waist, hip and thigh. Lower extremity muscle strength (Biodex dynamometer), grip 
strength, physical performance (expSPPB), and 400-m walk time will be measured. The MAT-sf and DSST will 
be administered. The CHAMPS Physical Activity Questionnaire, CES-D, SF-36, and FACIT questionnaires will 
be administered at the visit time permitting or by phone after the completion of the in-person visit. A series of 
CT scans (abdomen, hip and thigh) will be done. Participants will be given the 24-hr urine collection bottle and 
instructions for collecting urine to measure urinary nitrogen and creatinine for estimating dietary protein intake 
and for storage. Three 24-hr diet recalls will be administered by phone during the FV1/2 visit window. . 
Participants will schedule to drop off their 24-hour urine sample following their FV1/2 visit 
Monthly phone calls: Participants will be called once a month during the 12-mo follow-up to ensure those that 
are in the 18-mo HiProt are continuing to consume the protein supplement. All participants will be asked about 
any health changes, injuries, etc., that may have occurred and any events recorded using the Adverse Event 
Form.  
The 12-month follow-up visit (FV3) will take place following week 52 of the intervention. Participants will have 
their weight and blood pressure measured. Lower extremity muscle strength, grip strength, physical 
performance (expSPPB), and 400-m walk time will be measured. The CHAMPS Physical Activity 
Questionnaire, MAT-sf, CES-D, SF-36, and FACIT questionnaires will be administered. Medical history and 
current medications and dietary supplements will be reviewed.  
The 18-month follow-up visit (FV4/5) will take place following week 78 in the morning (after an 8 hour 
overnight fast). Participants will have their weight and blood pressure measured. Blood will be drawn for 
glucose, insulin, HbA1c, lipids, CMP and CBC, and storage. Participants will then be provided with a snack. 
Any changes to medical history and current medications and dietary supplements will be reviewed. Whole-
body, AP spine, and hip DXA scans will be done. The participant will have anthropometric measurements 
taken at their waist, hip and thigh. Lower extremity muscle strength (Biodex dynamometer), grip strength, 
physical performance (expSPPB), and 400-m walk time will be measured. The MAT-sf and DSST will be 
administered. The CHAMPS Physical Activity Questionnaire, CES-D, SF-36, and FACIT questionnaires will be 
administered at the visit time permitting or by phone after the completion of the in-person visit. A series of CT 
scans (abdomen, hip and thigh) will be done. Participants will be given their 24-hr urine collection bottle and 
instructions for collecting urine to measure urinary nitrogen and creatinine for estimating dietary protein intake 
and for storage ahead of their FV4/5 visit and asked to return it at their FV4/5 visit. Three 24-hr diet recalls will 
be administered by phone between during the FV4/5 visit window.  
After completion of all study visits, participants will be mailed a results packet and be informed of their 
randomization assignment. 
In the event of an abnormal laboratory value or test after randomization (e.g., eGFR), the participant will be 
asked to return to the clinic and provide an additional blood sample and/or have the test repeated. 

V08.02.22 
 
 
 
11 
Outcome Measures 
The nature, purpose, and risks of all procedures and protocols will be explained to each participant before 
obtaining written consent. All examiners will be trained in the standardized conduct of all assessments before 
data collection. Participants will be instructed to wear appropriate and comfortable clothing, and standardized 
written instructions will be provided prior to each study visit.   
Primary outcomes (assessed at baseline and at 6-, 12- and 18-month follow-up):  
Physical performance will be assessed using a modified version of the Short Physical Performance Battery 
(SPPB), a widely used assessment of lower extremity physical function which includes progressively more 
challenging standing balance tasks held for 10 seconds each (side-by-side, tandem and semi-tandem), the 
faster of two 4-m walks to assess usual gait speed, and time to complete 5 repeated chair stands.45  Each of 
the three performance measures is assigned a score ranging from 0 (inability to do the test) to 4 (the highest 
level of performance) and summed to create an SPPB summary score ranging from 0 (worst) to 12 (best). We 
will also administer the components of the expanded Short Physical Performance Battery (expSPPB) which 
was modified from the original SPPB to increase the holding time of the semi- and full-tandem stands to 30 
seconds and add a single leg stand and a narrow walk test of balance (walking at usual pace within lines of 
tape spaced 20 cm apart). The scoring system was developed to avoid ceiling effects observed with the SPPB 
battery.43 ExpSPPB scores are continuous and range from 0 to 4, with higher scores indicative of better 
performance.  
Maximal isokinetic knee extension and flexion strength will be measured using an isokinetic dynamometer 
(Biodex) at two speeds (60°/sec and 120°/sec) with the participant sitting and the hips and knee flexed at 90°.  
The dynamometer will be adjusted for each participant and all adjustments will be recorded to duplicate the 
position for subsequent assessments. Start and stop angles will be set at 90° and 30°. Strength is expressed in 
Newton-meters (Nm). Two trials will be done consisting of 4 repetitions each. Participants who have had a 
brain aneurysm, cerebral hemorrhage in the last 6 months, eye surgery (e.g., cataract surgery) within the past 
month, blood pressure >199/109 mmHg, bilateral knee replacement, or have difficulty bending or straightening 
both legs due to pain, arthritis, injury or some other condition will not be tested. The maximum knee extensor 
strength of the 4 repetitions from trial 2 for the dominant leg will be will be used in analyses unless unable to 
test the dominant leg (i.e., knee replacement) in which case the non-dominant leg will be used; the maximum 
knee extensor strength from the same leg will be used at 6- and 18-month follow-up. 
Grip strength will be measured twice in each hand using an isometric hydraulic hand dynamometer (Jamar, 
Bolingbrook, IL) and the mean value from the stronger hand used. Participants will be excluded from 
performing the test if they report hand-pain or recent hand or wrist surgery.   
The 400-meter walk is a walking-based test of exercise tolerance and aerobic fitness.43 Participants are 
instructed to complete the 400 m distance (on a flat indoor surface) as quickly as possible at a maintainable 
pace and the time to complete the walk is recorded in seconds. Encouragement is given in a standardized 
fashion every lap. The test will be terminated if the participant is unable to continue because of pain, fatigue, or 
other symptom. The test has excellent reproducibility (ICC=0.95),47 directly correlates with measured maximal 
aerobic fitness.48  
Secondary outcomes (assessed at baseline and at 6- and 18-month follow-up):  
Body composition. Fat and whole body and appendicular lean mass will be measured by dual-energy X-ray 
absorptiometry (DXA; Horizon A, Hologic Inc.). Whole body scans will be acquired with the participant supine 
and aligned with the scanner table. All scans will be performed and analyzed by our DXA technician who is 
certified by the International Society for Clinical Densitometry and has vast experience in body composition 
assessments in older adults. Daily quality control scans are obtained with Hologic’s phantom. Anthropometric 
measures of fat distribution will be assessed by circumference measurements of the waist, hip, and thigh using 
a tape measure. 
Thigh muscle volume and body fat distribution. Thigh muscle volume and inter-muscular adipose tissue 
(IMAT) and subcutaneous and visceral abdominal fat (SAT and VAT, respectively) will be measured by 
computed tomography (CT; Siemens SOMATOM Definition Flash 64-slice CT scanner,Siemens Medical 
Solutions USA, Inc., Malvern, PA). Participants will be positioned supine with the arms above the head and 
legs positioned flat. A 5-cm section of the thigh centered at the junction of the proximal and middle third of the 
femur will be used to quantify thigh muscle and adipose tissue. Thigh muscle attenuation (Hounsfield units), 

V08.02.22 
 
 
 
12 
excluding intermuscular fat, will also be assessed and used to quantify IMAT. CT slices within 15 mm centered 
at the L4-5 level will be selected to quantify SAT and VAT. Quantitative measures of thigh muscle and adipose 
tissue volume and CT attenuation values will be obtained with the GE Healthcare, Advantage Windows 4.2 
Volume Viewer (Waukesha, WI).  This software provides the ability to determine tissue-specific thresholds 
based on CT number (-190 to -30 Hounsfield units for fat) and volumetrically measure complex structures 
using semi-automated techniques such as edge attraction and volume rendering. The same CT reading 
technician will analyze all scans.  Daily calibration scans are obtained according to the manufacturer’s 
recommended procedure.  
Tertiary outcomes (assessed at baseline and at 6- and 18-month follow-up [Arterial Stiffness at baseline and 6-
month follow-up only]): 
Bone mineral density. DXA will be used to analyze bone mineral density (BMD) using specific scans of the 
anterior-posterior (AP) spine (L1-L4) and proximal femur.  
Volumetric bone mineral density, morphometry and strength. Helical CT scans of the lumbar spine and 
hip/pelvis will be used to measure volumetric bone mineral density, structure and strength of the lumbar spine 
(L1-L5), total hip and proximal femur. The scan coverage for the lumbar spine will be from the top of L1 
through the base of L5. The hip/pelvis scan will cover the region from the superior acetabulum to mid-femur. 
The mid-femur position will be defined as the midpoint between the superior aspect of the greater trochanter 
and the inferior aspect of the lateral condyle. To locate the mid-point, an anterior-posterior scout of the entire 
femur will be obtained. The same Mindways bone mineral phantom (Mindways Software, Inc., Austin, TX) will 
be imaged in every scan.   
Lipoprotein lipids. The lipoprotein lipid assays will be performed by LapCorp.  
Fasting glucose and insulin. Fasting glucose and insulin assays will be performed by LapCorp. An estimate 
of insulin sensitivity by the homeostasis model assessment (HOMA) score will be calculated with the formula: 
fasting plasma insulin (μU/ml) × glucose (mmol/l)/22.5.51 Glucose and insulin area under the curve will be 
determined using Tai’s model: ½×30×(y0min+2y30min+2y60min+2y90min+y120min), where y represents insulin or 
glucose values at the different time points.52  
Blood pressure will be measured in the right arm, using an automated sphygmomanometer, with the 
participant in a seated position after having rested quietly for 10-15 minutes. Systolic and diastolic blood 
pressure will be defined as the average of three repeated measures.  
Arterial Stiffness measures. The SphygmoCor XCEL system is an automated device that will be used to 
measure central pressure waveform and corresponding indices (i.e., central blood pressure, aortic 
augmentation index [AIx], and reflection magnitude) using pressure oscillations from an arm cuff and a 
validated, generalized transfer function.54-56 Aortic AIx is calculated as the ratio of augmentation pressure (i.e., 
the supplementary increase in systolic blood pressure due to early arterial wave reflections) to pulse pressure 
and is expressed as a percentage.57 Reflection magnitude is determined by separating the central pressure 
wave into its forward and backward components and is calculated as the ratio of the forward and backward 
wave amplitudes.58 
Descriptive variables and potential covariates: 
Perceived fatigue and maximal aerobic capacity (VO2max). Perceived fatigue will be assessed at a given 
workload. Participants will walk on a treadmill for 5 min at 0% grade at a fixed, comfortable speed of 2.0 mph, 
and heart rate and perceived exertion are recorded at midpoint and end. This test will be performed before the 
assessment of VO2max and serves as a warm-up to acclimate participants to the treadmill. VO2max will be 
determined on a motorized treadmill during a graded exercise test to exhaustion using a Ramp protocol to 
exclude those with exercise-induced ischemia at screening.44 Dr. Nicklas and Ms. Gordon (Master’s level 
Exercise Physiologist) have extensive experience measuring VO2max in older persons. Throughout the 
treadmill test, ventilatory and gas exchange responses will be measured on a breath-by-breath basis using a 
computerized system (Medical Graphics), which provides valid and reliable gas analysis.49;50 Data used to 
calculate the minute values will be collected for the entire 60-second period. If 60 seconds of data are not 
available for the last minute of the test, a minimum of 30 seconds of data must be collected and these data will 
be used to represent the last minute. Pre- and post-intervention testing will be conducted using the same 
treadmill and gas analysis system. VO2max will be expressed both as ml of oxygen uptake per kg of body 

V08.02.22 
 
 
 
13 
weight per minute (ml/kg bwt/min) and per kg of lean mass per minute (ml/kg LM/min). Two of the following 3 
criterion must be met for a test to be considered valid (or the test will be repeated): 1) plateau in oxygen 
consumption with increasing workload (< 200 ml/min); 2) respiratory exchange ratio ≥1.10; and 3) maximal HR 
within 90% of age-predicted maximal HR (208 – [0.7 x age]).   
Resting metabolic rate (RMR) will be measured at baseline for prescribing calorie deficits during the weight 
loss intervention and at 6 months for prescribing weight maintenance diets. The test will be conducted in the 
morning after an overnight fast using indirect calorimetry. Upon arrival, participants are asked to rest quietly for 
20-30 minutes before testing. Measurement of oxygen consumption and carbon dioxide production are 
collected continuously for up to 30 minutes and RMR is calculated using the Weir equation.59  
Demographic characteristics (age, sex, race, education, and socio-economic status) will be ascertained by 
questionnaire at the screening visit.  
A weight loss readiness questionnaire will assess participants’ motivation, commitment, and any barriers to 
attending the 6-month weight loss intervention at the screening visit. 
Medical information regarding co-morbidities will be ascertained at the screening visit. We will record 
medication and supplement use at baseline and at 6-, 12- and 18-month follow-up. Participants will be asked 
to bring in medications and dietary supplements for a "brown bag" review. The medication form will specifically 
ask about the use of protein supplements, anti-inflammatory agents, and sex steroids or corticosteroids. 
Cognitive function will be assessed at screening using the Montreal Cognitive Assessment (MoCA),31 
participants must score ≥22 to be eligible. We will also assess psychomotor speed, attention, and working 
memory using the Digit Symbol Substitution Test (DSST) at baseline and at 6- and 18-mo follow-up. 
Participants are given a series of numbered symbols and then asked to draw the appropriate symbols below a 
list of random numbers. The score is the number of correctly made matches in 2 minutes (120 seconds).  
Initial body weight and change in body weight: Participants will be weighed, without shoes or heavy 
clothing, on a standard, calibrated scale weekly during the 6-month weight loss intervention and at the 
baseline, 6-, 12- and 18-month follow-up visits.   
Compliance: During the 6-month weight loss phase, participants will be asked to track their daily intake of all 
foods and beverages in a log book provided to them and their log books reviewed at each session. 
Attendance at group and individual diet and exercise sessions and protein supplement usage during the 
study will also be tracked.  
Physical activity: Physical activity will be assessed by self-report at baseline, and at 6-, 12- and 18-month 
follow-up using the CHAMPS physical activity questionnaire.32  
Dietary intake: Three 24-hr dietary recalls (two week days and one weekend day) will be performed on all 
participants at baseline, and at 6- and 18-month follow-up. Experienced registered dietitians trained in 24-hr 
dietary recall interviews from the WFSM Clinical Research Unit (CRU) who are not involved with the diet 
intervention will conduct the 24-hour dietary recalls. Data will be collected and analyzed using the Nutrition 
Data System for Research (NDSR) software developed and supported by the Nutrition Coordinating Center at 
the University of Minnesota. The NDSR database is updated annually to reflect marketplace changes and new 
analytic data. The program also uses the multiple pass approach, providing interview prompts to guide and 
standardize data entry, thereby providing multiple opportunities for the participant to recall food intake and 
improve data accuracy.  A validated visual portion guide will be provided to the participant for use in 
determining accurate portion sizes. During the baseline visit, a CRU dietitian will obtain the first 24-hour recall 
in person, and this interview will be used to introduce and train the participant for the procedures used in 
subsequent 24-hr dietary recalls done by phone. The remaining 24-hr dietary recalls will be conducted over the 
phone (taking approximately 20-40 minutes each) by placing unscheduled telephone calls to participants. 
When contacted by the staff, participants will be asked to recall and report their dietary intake from the 
previous day’s 24-hour period.  
Urinary nitrogen excretion, a biomarker of dietary protein intake,60 will be used to verify protein intake 
between groups. Participants will be asked to collect urine over a 24-hr period at baseline, 6- and 18-month 
follow-up. 

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14 
Depressed mood will be assessed using the 20-item Center for Epidemiologic Study Depression Scale (CES-
D)33 at screening and at 6-, 12- and 18-mo follow-up. Individuals who score ≥16 at screening will be evaluated 
by the study team to determine if appropriate to participate in the study (e.g., participant did not exhibit 
depressed mood or a lack of interest in activities during study visit). In the event that a participant’s 6-, 12- or 
18-mo CES-D score increases and is above the threshold indicating that an individual is at risk for clinical 
depression (CES-D score ≥16) and the study team noted that the participant is exhibiting depressed mood or a 
loss of interest in daily activities, a letter will be sent to the participant advising them to follow-up with their 
PCP. 
The Short Form-36 Health Survey (SF-36) will be used as a measure of quality of life and assessed at 
baseline and at 6-, 12- and 18-mo follow-up. The SF-36 measures 8 health domains: physical functioning, role 
limitations because of physical health problems, bodily pain, general health perceptions, vitality, social 
functioning, role limitations because of emotional problems, and general mental health.61 Scores for each 
health domain subscale range from 0 to 100, with higher scores indicating better functioning or well-being. 
Activities of daily living will be assessed using the Mobility Assessment Tool – Short Form (MAT-sf) at 
baseline, 6-, 12- and 18-month follow-up. This is a novel, computerized tool for self-assessment of functional 
performance designed to reduce bias from factors such as age, gender and body image.62 
Fatigue measures. The FACIT Fatigue Scale will be administered at baseline, 6-, 12- and 18-month follow-up. 
Adverse events will be assessed by asking participants at each assessment and intervention visit and on 
monthly phone calls during the 12-mo follow-up to report any health changes, injuries, etc., and these will be 
recorded using the Adverse Event Form.  
Analytical Plan 
Sample size: The sample size chosen for this study (n=225; 75/group) will provide ≥80% power (α=0.05) to 
detect clinically meaningful effects of a higher protein / lower CHO diet during weight loss and follow-up on 
physical function (with estimated retention of 80% or 60 completers per group; see Table 3). Baseline standard 
deviations and pre-post correlations used for power analyses were calculated using data from obese older 
adults from our OPTIMA study11 – a weight loss study with the same inclusion/exclusion criteria as the 
proposed study. Standard deviations were assumed to be similar between groups. The mean group 
differences we can detect correspond to an absolute change from average baseline values of these measures 
in obese older adults of 0.2 points for the expSPPB and 12.6 Nm for muscle strength. Differences of 0.12 
points and 0.22 points on the expSPPB have been shown to be small and large clinically meaningful 
differences, respectively.63  Even with a conservative drop-out rate of 20%, 60 participants/group provides 
≥80% power (overall α=0.05, 2-sided tests for 3 comparisons) to detect between group differences for primary 
and secondary outcomes over 18 months.  In response to our NIA-appointed DSMB, we have recalculated the 
power based on the actual number of participants enrolled (total n=187 or ~50 per group after accounting for 
the 20% drop-out rate) using the same baseline standard deviations and pre-post correlations from obese 
older adults in our OPTIMA study noted above. 
Table 3.  Power to detect significant differences in mean change between groups with ANCOVA (α=0.05 after Bonferroni correction 
for each outcome; 2-sided test), assuming similar SD between groups 
Outcome  
measure 
Group Comparison 
Pre-Post 
Correlation 
Standard 
Deviation
(SD) 
Mean 
group 
difference 
Effect 
Size 
(diff/SD) 
Power for Analysis of Covariance  
(n per group) 
n=60 
n=65 
n=75 
n=50 
ExpSPPB 
(points) 
 
RecProt vs. 6mo HiProt 
0.72 
0.5 
0.20 
0.41 
80 
84 
89 
71 
6mo vs. 18mo HiProt 
 
 
0.20 
0.41 
80 
84 
89 
71 
RecProt vs. 18mo HiProt 
 
 
0.41 
0.82 
>99 
>99 
>99 
>99 
Knee 
extensor 
strength (Nm) 
RecProt vs. 6mo HiProt 
0.77 
32.8 
12.6 
0.38 
81 
84 
90 
72 
6mo vs. 18mo HiProt 
 
 
12.6 
0.38 
81 
84 
90 
72 
RecProt vs. 18mo HiProt 
 
 
25.3 
0.77 
>99 
>99 
>99 
>99 
Lean mass 
(Kg) 
 
RecProt vs. 6mo HiProt 
0.99 
12.0 
1.1 
0.09 
86 
89 
93 
78 
6mo vs. 18mo HiProt 
 
 
1.1 
0.09 
86 
89 
93 
78 
RecProt vs. 18mo HiProt 
 
 
2.2 
0.18 
>99 
>99 
>99 
>99 
 
 
 
 
 
 
 
 
 
 
 

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15 
Table 3.  Power to detect significant differences in mean change between groups with ANCOVA (α=0.05 after Bonferroni correction 
for each outcome; 2-sided test), assuming similar SD between groups 
Outcome  
measure 
Group Comparison 
Pre-Post 
Correlation 
Standard 
Deviation
(SD) 
Mean 
group 
difference 
Effect 
Size 
(diff/SD) 
Power for Analysis of Covariance  
(n per group) 
n=60 
n=65 
n=75 
n=50 
IMAT (cm3) 
RecProt vs. 6mo HiProt 
0.98 
31.5 
4.1 
0.13 
88 
91 
94 
80 
6mo vs. 18mo HiProt 
 
 
4.1 
0.13 
88 
91 
94 
80 
RecProt vs. 18mo HiProt 
 
 
8.2 
0.26 
>99 
>99 
>99 
>99 
 
Database management: To facilitate data transfer and preserve records that can be audited, we will use hard 
copy forms collection. Data will be entered into web-based forms on a continuous basis as collected and 
analysis datasets will be stored in SAS databases. All data will undergo range checks at the time of data entry 
and will be examined periodically by histograms and bivariate scatterplots to check for inconsistencies, unusual 
data needing further verification, and outliers. Plots of longitudinal observations will be used to inspect for 
unusual changes that need to be verified against source documents. Regression diagnostics and exploratory 
analyses will be performed to find appropriate transformations of variables if needed.  
Data analysis: Initial analyses will follow the “intent-to-treat” principle in which data from all randomized 
participants are analyzed according to their randomized groups. Analysis of covariance (ANCOVA) will be used 
for both primary and secondary aims to compare the group effect on change in expSPPB and muscle strength 
(primary aim) and weight loss maintenance, total and appendicular lean mass and fat-to-lean mass ratio, and 
thigh muscle volume and inter-muscular adipose tissue (IMAT) (secondary aim) at 18 months. Group contrasts 
will be constructed and tested within each model. ANCOVA will also be used to compare physical function, 
weight and body composition at 6 months. To explore whether differences in lean mass mediate the effect of a 
higher protein diet during and subsequent to weight loss on physical function, change in lean mass will be 
added to the ANCOVA models to assess whether the intervention effect is reduced or eliminated as an ad hoc 
mediation analysis. To compare the exploratory outcomes (bone mineral density and cardiometabolic risk 
factors) at 6 and 18 months, ANCOVA with repeated measurements will be used, and correlations between 6 
and 18 months taken into account using a compound symmetry covariance structure. All models will be 
adjusted for the baseline measure, age, gender, and race.  
Factors that may influence the measured responses to the interventions (e.g., initial body weight or 
composition, changes in weight, physical activity, etc.) will be evaluated and possibly included in secondary 
analyses. We will also consider conducting analyses adjusted for intervention process measures (such as 
dietary intake, average attendance at diet and exercise sessions, and protein intake/urinary nitrogen) to 
examine the extent to which these contribute to the outcomes. 
Planned sensitivity analyses to assess the impact of COVID19: Because of the potential impact of COVID19 
on intervention delivery and follow-up assessments, we plan to conduct several sensitivity analyses: 
1) Exclude Wave 13 and 14 participants (n=26) whose intervention was interrupted by the COVID19 research 
pause on March 13, 2020, and who completed the last ~2 and ~4 months, respectively, of the exercise 
portion of the 6-month intervention on their own while attending the diet portion of the intervention remotely 
(via phone). 
2) Exclude Wave 13-17 participants (n=63) whose intervention was interrupted by COVID19 (Waves 13 and 
14; see above) or whose intervention was modified from earlier waves to accommodate safety measures 
put in place due to COVID19 (Waves 15-17; center-based exercise reduced from 3x/week to 1-2x/week 
after week 6 to allow for physical distancing). 
3) Exclude Wave 7 participants (n=15) and participants from Wave 6 (n=5) whose 18-month visits were 2-3 
months out of window due to the COVID19 research pause and phased restart of in-person research. 
Missing data considerations:  We will document withdrawal and compliance in detail to permit the formulation 
of reasonable assumptions regarding missing observations. If it is determined that our outcomes are missing at 
random (MAR), we will attempt to identify baseline covariates that predict attrition and use these covariates to 
impute missing data based on multiple imputations (MI) and draw inferences based on MI. If it is determined 
that our outcomes are not missing at random (MNAR), sensitivity analyses MNAR assumptions will be carried 
out using MI following the recommendation of Molenberghs and Kenward.64  
 
 

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Human Subjects Protection 
Subject Recruitment Methods:  We will recruit individuals using community-based recruitment strategies 
including newspaper ads and mass mailings. We will also advertise in the Aging Center’s VITAL newsletter 
and participate in community outreach events.  
Informed Consent:  Written informed consent will be obtained from each study participant. The informed 
consent process will follow the procedures of the WFU Institutional Review Board. The study interviewers will 
explain the purpose, methods and extent of the study to prospective participants. The potential participant is 
asked to read the informed consent form and ask questions. The form will be written in simple easy to 
understand language. We require study staff to review all of the key aspects of the study verbally with the 
potential participants. Staff is then required to question potential participants to ascertain whether s/he has 
understood the information. Potential participants who are illiterate or have impaired vision must have the 
consent read to them, followed by review of the checklist, opportunity for questions, and discussion. This 
process will take place in a quiet, private room. A copy of the signed and dated consent form will be given to 
participants, and the original document will be placed in subjects’ individual study files, which will be stored in a 
secure location. In compliance with the Health Insurance Portability and Accountability Act (HIPAA) and the 
Standards for Privacy of Individually Identifiable Health Information of the Department of Health and Human 
Services, we will access personal health information only after obtaining informed consent. 
Potential Study Risks:  There are inherent potential risks to human subjects who participate in any research 
study and the potential risks to study participants in this study are listed below. Any injuries or illnesses (severe 
adverse events) during the course of a participant’s enrollment in the study are monitored regularly as 
described below in the Data Safety Monitoring Plan.  
1) Physical Function Testing.  There is a small risk of injury during the muscle strength testing and 
physical performance testing, such as muscle strains or pulls, falls, or joint injury. However, these tests 
have been performed in large study populations with no significant adverse events reported. Risks will 
be minimized by having experienced/trained staff conducting these assessments. A warm-up and range 
of motion practice will be conducted before maximal strength testing. In addition, if a participant reports 
pain, dizziness, lightheadedness or other medical problem during the test, the test will be terminated.   
2) Radiation Exposure.  There are risks with exposure to radiation from the whole body, anterior-posterior 
spine, and proximal femur DXA scans and chest, abdominal, pelvic and thigh CT scans. Exposure to 
radiation during the DXA (1-10 mRem each) and CT (137-255 mRem each) scans for a total of 1860 
mRem of radiation exposure from the DXA and CT scans over the three time points during the 18 
months of the study which is lower than the maximal permissible dose for occupationally exposed 
individuals. The potential long-term risk from these radiation doses is uncertain, but these doses have 
never been associated with any definite adverse effects. Thus, the risk to participants, if any, is 
estimated to be slight.     
3) Blood Draw.  Participants may experience temporary pain, bruising, bleeding and/or a small  
risk of infection, fainting or dizziness during the blood sample collection process. Blood will be drawn 
only by trained and experienced phlebotomists who will minimize the discomfort as much as possible 
and will use good clinical practice procedures to reduce risk of infection. 
4) Exercise testing is a common procedure with minimal risks, but the test is monitored by an exercise 
physiologist and will be stopped if problems occur. These include fainting, dizziness, chest pain, irregular 
heartbeats, or a heart attack, although the latter is extremely rare (<1 death in 10,000 tests) in people 
with no history of heart disease. Blood pressure, heart rate and rhythm, and breathing will be closely and 
constantly monitored. 
5) Resting Metabolic Rate.  Some participants begin to feel claustrophobic when the clear plastic mask is 
placed over their mouth. Should this occur we will remove the mask and stop the test. 
6) Risks of weight loss.  There are no known serious risks associated with using caloric restriction to lose 
weight. Changes in usual bowel function (diarrhea and/or constipation) may occur when beginning the 
new diet due to differences between the weight loss diet and the participant’s usual diet. Under 
conditions of rapid weight loss (more than 5 pounds per week), there is a very small chance of 
developing gallbladder disease. Risks at any age include the concomitant loss of lean (muscle and bone) 

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tissue along with fat mass loss. However, the clinical impact of this muscle and bone loss is not known. 
In older adults, this decrease in muscle mass and bone density with weight loss may result in increased 
risk for sarcopenia and/or osteopenia/osteoporosis. Since most older adults with obesity have a higher 
muscle mass and bone density, this risk is mitigated by excluding individuals who are non-obese and 
less likely to benefit from weight loss and excluding those with an increased risk for sarcopenia or 
osteoporosis. Individuals with T-score < -2.5 on hip or spine DXA scan at screening will be excluded. 
Procedures to minimize loss of muscle and bone during the weight loss intervention include prescription 
of a caloric deficit that does not result in excessively rapid weight loss (e.g., >2 lb/week), inclusion of 
increased weight-bearing activity (exercise intervention), and incorporating dietary recommendations and 
meal plans that include the current recommended dietary allowance (RDA) for protein (0.8 g/kg body 
weight/d) and counseling to consume, either from food or dietary supplements, 1200 mg/d of calcium and 
800 IU/d of vitamin D.   
7) The risks of the higher protein diet intervention are minimal, but may include a worsening of existing 
kidney problems. However, we will not enroll individuals with evidence of preexisting impaired kidney 
function for whom a diet with a higher protein level is contraindicated (i.e., eGFR<30 based on serum 
creatinine, age, gender, and race).   
8) Exercise Program.  The risks of the exercise program are minimal, but may include musculoskeletal 
complications and muscle soreness in the early phases of the training. This will be minimized since all 
exercise sessions during the first 6 weeks of the 6-month weight loss intervention and up to 2 sessions 
per week for the remainder of the 6-month weight loss intervention will be center-based and supervised 
by trained exercise interventionists, who will instruct participants in the proper exercise techniques and 
proper footwear (unless conditions arise that necessitate a transition to all home-based exercise – e.g., 
increased community spread of COVID19, new restrictions on in-person research are put in place, 
and/or if stay at home orders are put back in place). Procedures to minimize injury include warm-up and 
cool-down activities that include large muscle movements and stretching. Participants will begin with an 
easier exercise stimulus and will gradually increase in intensity over several weeks. Exercise 
physiologists, trained in cardiac life support, will supervise all center-based exercise sessions and 
practice codes are conducted quarterly. Tips on how to safely exercise in a home-based setting will be 
reviewed by the exercise interventionist during the first 6 weeks of center-based exercise.  
Confidentiality and Privacy 
All data are obtained for research purposes only. Confidentiality of data is maintained by using research 
identification numbers which uniquely identify each individual. Data will be used only in aggregate and no 
identifying characteristics of individuals will be published or presented. Results of testing will be sent to 
participants’ private physicians if participants agree to this. Confidentiality of data is maintained by using 
research identification numbers which uniquely identify each individual. The information collected from 
participants in this study has a low potential for abuse because the data does not address sensitive issues. 
Nevertheless, appropriate measures are taken to prevent unauthorized use of study information. Data other 
than demographic information do not use names as an identifier – the research ID number is used. The 
research records are kept in locked cabinets in the Geriatric Research Clinic. The files matching the 
participants' names and demographic information with the research ID numbers are kept in a separate room 
and locked file that uses a different key from that of all other files. A web-based data entry system will be 
created for this study which will allow access only to study staff (user id and password required to access). 
Information linking IDs to individuals is kept on a secure, password-protected server to which only authorized 
study personnel will have access. Blinded staff will not have access to blinded study information on the web-
based database. Computer files are stored on file servers, which are backed up each night and stored for easy 
retrieval in case of emergency. Files may not be obtained from the research unit by persons other than 
research personnel, who are asked to sign a document agreeing to maintain the confidentiality of the 
information. After the study is completed, the local data will be stored with other completed research studies in 
a secured storage area. 
Data and Safety Monitoring 
The PI, along with the study physician, will be responsible for the overall monitoring of the data and safety of 
study participants. In addition, all clinical intervention studies conducted by Aging Center investigators are 
monitored by the WFU Claude D. Pepper Older Americans Independence Center’s NIH-approved Data Safety 

V08.02.22 
 
 
 
18 
Monitoring Board (DSMB). Finally, the external NIA-appointed Data and Safety Monitoring Board (DSMB) will 
have overall responsibility of independently monitoring all aspects of the study, including those that require 
access to any un-blinded data. This committee acts in an advisory capacity to the NIA to monitor participant 
safety, evaluate the progress of the study, review procedures for maintaining the confidentiality of data, and the 
quality of data collection, management, and analyses. The DSMB will have access to all study data, 
documents and progress information, and will be notified of all changes that are made to the protocol.  
The principal investigator will be responsible for the overall monitoring of the data and safety of study 
participants. The principal investigator will be assisted by other members of the study staff. The PI, co-
investigators, and the biostatistician will meet regularly to review study progress and to examine reports of 
adverse incidents as well as participant recruitment and follow-up. All serious adverse events will be reported to 
the Institutional Review Board (IRB) and if the event occurred as a direct result of participation in this study, an 
amendment will be made to the consent form and the PI will request new IRB approval. The PI and the study 
physician (Dr. Lyles) will meet quarterly, or as needed, to review all reported events. In addition, participant 
safety in all Wake Forest Pepper Center supported studies is monitored by a Data and Safety Monitoring 
Committee (DSMC). It reviews study recruitment, drop-outs, protocol changes, losses to follow-up, and adverse 
events every 6 months.   
Adverse events include any event that occurs during the course of the study that results in a participant 
suffering physical or mental injury, pain or suffering. Adverse events can be major, such as a subject who 
suffers cardiac arrest during neuromuscular function testing, or minor such as a subject pulling a muscle during 
neuromuscular function testing. This includes any events occurring while a subject is enrolled in the study, even 
if the event did not occur while s/he was actively participating in the activities called for in the research protocol. 
Deviations from the study’s protocol are also considered an adverse, unexpected, or notable event and will be 
reported to the PI. 
Both major and minor events will be reported using the study’s Adverse, Unexpected, or Notable Event 
Reporting Form. Any major related and unexpected event, i.e., any serious injury or life-threatening event, will 
be reported immediately (within 24 hours of notification) to the WFSM Institutional Review Board (IRB) right 
after completing any and all actions that are necessary to protect the subject’s health and safety. The SAE will 
also be reported within 24 hours to the NIA and Chair of the DSMB via a verbal phone call, or by a report 
submitted by fax or email. Minor events will be reported within seven days. A description of the event, and the 
date and location of the event will be recorded on this form, which will be kept in the subject’s research file. 
Reporting of Unanticipated Problems, Adverse Events or Deviations 
Any unanticipated problems, serious related and unexpected adverse events, deviations or protocol changes 
will be promptly reported by the principal investigator or designated member of the research team to the IRB 
and sponsor or appropriate government agency if appropriate. 
Use of biological samples by other investigators 
Biological samples may be used by investigators other than the investigators of the current study. The use will 
be limited to non-commercial purposes. The names and other personal identifiers of the study participants will 
not be sent to any recipients of the blood samples. 
Storage and disposal of biological material 
Blood and urine samples will be stored at Wake Forest University Medical Center for up to twenty years after 
the end of the trial at which time the samples will be destroyed. Biological specimens will be stored in locked -
70oC alarmed freezers located in a locked room. The Wake Forest Pepper Center Integrative Biology Core lab 
coordinator (Karin Murphy) and the Core Leader (Dr. Nicklas) have access to the keys of the freezers. All the 
specimens will have numerical study IDs with no personal identifiers of the participants. These are stored 
under the Pepper Center Tissue Repository (IRB#1219). 
 
 
 

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