Trial of Vitamin D Supplementation and Neuromuscular Function in Older Adults
Phase: NA
Conditions: Neuromuscular Function
Sponsor: Wake Forest University Health Sciences

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Grant Study Title: Trial of vitamin D supplementation and neuromuscular function in older adults  
Working Study Title: EVIDENCE - Exploring VItamin D’s Effects on NeuromusCular Endpoints Study 
 
Principal Investigator, Co-investigator(s): Denise K. Houston (PI), Steve Kritchevsky, Osvaldo Delbono, 
Janet Tooze, Tony Marsh, Anthony Molina, Jamehl Demons, and Claudia Campos 
 
Sponsor or funding source: NIH/NIA and The Office of Dietary Supplements 
 
Background, Rationale and Context 
The scientific community disagrees on the amount of vitamin D required for optimal health. In 2011, the 
Institute of Medicine (IOM) concluded that while scientific evidence supports a role of vitamin D for bone 
health, compelling evidence was lacking for non-bone health outcomes, primarily due to the paucity of 
randomized trials.1 The IOM also concluded that 25-hydroxyvitamin D (25[OH]D) concentrations ≥20 ng/mL (50 
nmol/L) were sufficient for bone and overall health. However, many vitamin D experts, citing a large volume of 
observational research, maintain that 25(OH)D concentrations ≥30 ng/mL (75 nmol/L) are beneficial for non-
bone health outcomes such as neuromuscular function.2-4 The resolution of this controversy is vital given that 
almost 20 million older adults have 25(OH)D concentrations between 20 and 30 ng/mL.5 If increasing 25(OH)D 
concentrations above 30 ng/mL is beneficial, it would have an enormous impact on improving the health of 
approximately one-half of the older U.S. adult population. To resolve these controversies, randomized 
controlled trials are needed to both establish non-bone health benefits of remediating low 25(OH)D 
concentrations and determine whether increasing 25(OH)D concentrations from ≥20 to ≥30 ng/mL is beneficial. 
Growing evidence indicates that vitamin D’s effect on reducing falls is mediated by improvements in 
neuromuscular function.6-8 We and others have shown associations between 25(OH)D concentrations and 
muscle strength and physical performance measures associated with fall risk (e.g., gait, balance) in 
observational studies.6,9-13 Yet trials of vitamin D supplementation on changes in muscle strength and physical 
performance are equivocal,14 likely due to inadequate trial duration, small sample sizes, insufficient vitamin D 
dose, and sample heterogeneity. Moreover, vitamin D’s effects on the mechanisms underlying neuromuscular 
function are not well understood. Determining whether increasing 25(OH)D concentrations to ≥30 ng/mL will 
improve neuromuscular deficits that are risk factors for falls and elucidating the underlying physiological 
mechanisms linking vitamin D and neuromuscular function could change clinical practice by providing evidence 
to guide vitamin D supplementation recommendations for neuromuscular-related outcomes in older adults. 
Objectives 
To conduct a 12-month, double-blind randomized placebo controlled trial in 136 older (65-89 yrs) men and 
women with initial 25(OH)D concentrations of 18-<30 ng/mL to determine the effect of increasing 25(OH)D 
concentrations to ≥30 ng/mL through vitamin D3 supplementation on 1) change in neuromuscular functions that 
are established risk-factors for falls in older adults; and 2) changes in the underlying physiological mechanisms 
over 4 months in a subset of 36 randomly selected participants. Participants will be randomized to 2000 IU/d of 
vitamin D3 (a dose expected to increase 25(OH)D concentrations by ~14 ng/mL) or placebo. 
Hypothesis 1: Compared to participants randomized to placebo, participants randomized to vitamin D3 will 
have increased lower extremity muscle power and strength, increased physical performance, decreased 
postural sway, and reduced rate of falls over 12 months. 
Hypothesis 2: Compared to participants randomized to placebo, participants randomized to vitamin D3 will 
have increased type II muscle fibers, muscle fiber force, and myofiber regeneration, and decreased muscle 
fiber denervation over 4 months.  
Lower extremity muscle strength and power, physical performance, and postural sway will be assessed at 
baseline, 4 months and 12 months and falls assessed monthly. Muscle biopsies of the vastus lateralis will be 
taken at baseline and 4 months to assess muscle fiber type, contractility, and denervation, and number and 
differentiation stage of satellite cells. The primary outcomes are change in lower extremity muscle power 
(Hypothesis 1) and change in percentage of type II (fast-twitch) muscle fibers (Hypothesis 2). Secondary 
outcomes include change in lower extremity muscle strength, physical performance, and postural sway 
(Hypothesis 1) and muscle fiber contractility, muscle fiber denervation, and number and differentiation of 
satellite cells (Hypothesis 2). Falls will also be assessed as an exploratory outcome (Hypothesis 1). 
 
 

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Methods and Measures 
Experimental Overview/Study Design 
 
This study is a 12-month, double-blind randomized placebo controlled trial in 136 older (65-89 yrs) men 
and women with initial 25(OH)D concentrations of 18-<30 ng/mL to determine the effect of increasing 25(OH)D 
concentrations to ≥30 ng/mL through vitamin D3 supplementation on 1) change in neuromuscular functions that 
are established risk-factors for falls in older adults and 2) changes in the underlying physiological mechanisms 
over 4 months in a subset of 36 randomly selected participants. Participants will be randomized to vitamin D3 
(2000 IU/d) or matching placebo in a 1:1 ratio stratified by gender and race.  
Recruitment of study participants 
 
We plan to recruit and randomize a total of 136 older men and women with baseline 25(OH)D 
concentrations of 18-<30 ng/mL to 2000 IU/d vitamin D3 or matching placebo for 12 months in a 1:1 ratio 
stratified by gender and race. In addition, a subset of participants that are eligible to receive the muscle biopsy, 
i.e. those not taking anti-coagulants, will be randomly selected in a 1:1 ratio stratified by gender and race to be 
included in the 4 month muscle biopsy mechanistic substudy. We will stop recruitment into the muscle biopsy 
substudy when we can ensure that at least 32 participants will complete the pre- and post-muscle biopsy. Our 
power analyses show that 125 persons total (assuming 90% retention) will yield >80% power to detect 
between group differences of a 10% relative increase in lower extremity muscle power and 16 persons/group 
(assuming 32 completers) will yield >80% power to detect a 25% difference in the relative increase in type II 
muscle fibers between groups in the muscle biopsy substudy. We plan to recruit, screen, and enroll these 
individuals continuously at the rate of approximately 5-6/mo during years 1-4 of the study. We will target men 
and women aged 65 to 89 years who live in the surrounding area via media advertisements (newspaper), 
targeted mass mailings, the VITAL newsletter, and community outreach events. We will also target age-eligible 
patients being seen at the Downtown Health Plaza (DHP) via posters, flyers, targeted mass mailing, and 
approaching age-eligible patients following their scheduled DHP care provider visit to see if they would be 
interested in learning more about the study. 
Participant screening and randomization 
Telephone screening. All prospective participants will initially be screened via telephone to determine general 
eligibility. They will be asked about their age, medical history, and current medications and dietary 
supplements. All participants must meet the inclusion/exclusion criteria in Table 1. These selection criteria will 
eliminate participants with conditions that may affect their ability to safely perform the neuromuscular function 
tests, consume vitamin D supplements, or undergo a muscle biopsy. The telephone screen will be 
administered in person to interested DHP patients following their scheduled DHP care provider visit if time 
allows by the DHP recruitment coordinator; otherwise, it will be administered over the phone. 
Table 1. Inclusion/Exclusion Criteria 
Inclusion Criteria 
Exclusion Criteria
Age ≥65 to <90 years 
Serious or uncontrolled chronic disease including: insulin-dependent diabetes; cancer 
requiring treatment in past year, except non-melanoma skin cancers; acute coronary event 
(within the last 6 months), uncontrolled angina, heart failure (stage 3-4), PAD, or stroke 
(within the last 6 months); uncontrolled hypertension (BP>200/110 mmHg); chronic 
respiratory disease requiring the use of oxygen; uncontrolled endocrine/metabolic disease; 
neurological (e.g., Parkinson’s disease) or hematological disease; liver or renal dysfunction 
(eGFR <45 mL/min/1.73m2); and musculoskeletal impairments severe enough to preclude 
functional testing 
SPPB <10 if chair stand score is <3 
or <10 if chair stand score is a 4 
Evidence of impaired cognitive function (MoCA <18) 
Initial serum 25(OH)D concentration 
of 18 to <30 ng/mL 
Taking prescription vitamin D2  or taking >1000 IU/day of vitamin D3 (from all sources); 
taking an oral corticosteriod (i.e., prednisone at 7.5mg/d for 3 mos or equivalent); taking 
hormone replacement therapy 
Not dependent on a walker 
Inability or contraindications to consume daily vitamin D supplements (e.g., hypercalcemia, 
sarcoidosis, history of kidney stones in last 5 years) 
Willing to provide informed consent 
and adhere to the protocol 
Knee or hip surgery within the last 6 months or planned knee or hip surgery within the next 
year 
Not involved in another behavioral, 
exercise, or investigational drug 
intervention study 
Unwillingness to undergo a muscle biopsy  

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Table 1. Inclusion/Exclusion Criteria 
Inclusion Criteria 
Exclusion Criteria
Self- reported physical performance 
difficulty 
Weight loss of ≥5% or more in the past 3 months  
 
BMI >40 kg/m2 
 
Eye surgery (e.g., cataract surgery) within the past month or planned within the next month 
 
If the PI feels the participant is unlikely to follow the protocol 
 
Screening, consent and randomization. Individuals who pass the telephone screening will be scheduled for 
a screening visit (SV) in the Geriatric Research Clinic (GRC) in the Sticht Center on Aging. Before any data 
collection, all participants will provide written informed consent and complete a HIPAA authorization form in 
accordance with the Wake Forest School of Medicine Institutional Review Board policies. DHP patients who 
pass the in-person telephone screen will be given the option of completing a prescreen visit (PSV; informed 
consent, MoCA, SPPB, and weight and height for BMI; see below) with the DHP recruitment coordinator at the 
DHP after their scheduled DHP care provider visit if time allows. If they are eligible following the prescreen 
visit, they will be scheduled for the full screening visit at the GRC.  
Individuals who meet all eligibility criteria and provide informed consent will be invited back for the first 
baseline visit 1 (BV1). At the end of BV1, participants will be randomized to participate in the muscle biopsy 
substudy (those who are not on anti-coagulants) using a web-based randomization scheme (developed by Dr. 
Tooze) with blocking by gender and race with a goal of achieving n=32 pre- and post-muscle biopsy samples. 
At the end of baseline visit 2 (BV2), participants will be randomized to one of the two study interventions 
(vitamin D3 or placebo) using a web-based randomization scheme (developed by Dr. Tooze) with blocking by 
gender, race, and muscle biopsy subgroup. Block size and the randomization sequences will be unknown to 
the research staff. Muscle biopsy and intervention assignment will be generated using a program that the study 
coordinator will access from her PC through the study website.   
Schedule and organization of assessment visits 
 
All assessments will be conducted during 5-6 visits according to the timeline in Table 2: 1 screening visit; 2 
baseline visits; 1 or 2 (participants undergoing a muscle biopsy only) 4-month mid-intervention follow-up visits; 
and 1 12-month post-intervention follow-up visit. Assessments will take place in the Geriatric Clinical Research 
Unit, the Geriatric Research Clinic, or the Downtown Health Plaza (prescreen visit only).  
Table 2. Assessment timeline                                               Visit code 
SV 
BV1 
BV2 
On 
trial 
4FV1 
4FV2 
12FV 
Activity/assessment                                                       Week number 
-3 
-1 
0 
16 
17 
52 
Informed consent, review inclusion/exclusion criteria, demographics, 
weight*, height*, BP and pulse, fasting screening blood draw (metabolic 
blood panel and 25(OH)D), short physical performance battery 
(SPPB)*, cognitive screen (MoCA)*, medical history, 
medication/supplement review 
X 
 
 
 
 
 
 
Lower extremity muscle power, knee extensor and flexor strength,  
expanded SPPB, timed up and go, 20-m walk test (usual and fast), 
postural sway, gait velocity, 4 stair climb, grip strength, CHAMPS 
Physical Activity Questionnaire, arthritis and joint pain questionnaire, 
review changes to medical history and medications/supplements 
 
X 
 
 
X 
 
X 
Fasting blood draw (25(OH)D, 1,25(OH)D, storage), MAT-sf, sun 
exposure questionnaire, calcium and vitamin D diet questionnaire (done 
only at BV2) 
BP, height, weight, and a fasting metabolic panel will also be done at 
4FV and 12FV 
Fasting 25(OH)D (LabCorp) will also be done at 4FV and 12FV  to 
determine response to vitamin D supplements  
 
 
X 
 
X 
 
X 
CES-D, DSST. The MoCA will also be repeated at 12FV 
 
 
X 
 
 
 
X 
Muscle biopsy (subsample only) 
 
 
X 
 
 
X 
 
Randomization 
 
 
X 
 
 
 
 
Monthly supplement adherence and falls assessment calendars 
 
 
 
X 
 
 
 
SV = screening visit; BV = baseline visit; FV = follow-up visit; * See PSV below.  
 
The screening visit (SV) will be conducted in the morning following an 8-hour fast for measurement of 
25(OH)D concentration and a metabolic panel for screening purposes. After vitals (blood pressure, pulse, 

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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 short physical performance battery (SPPB)15 will be administered and 
scored. If still eligible, participants will then undergo a cognitive screen (assessed using the Montreal Cognitive 
Assessment, MoCA16) and a medical history and review of medications and dietary supplements. A prescreen 
visit (PSV)* may be scheduled to assess eligibility on the SPPB and the MoCA. If this occurs, the participant 
will sign consent at the PSV and then weight and height will be obtained to calculate BMI and the SPPB and 
MoCA will be administered. If the participant qualifies, they will then be scheduled for the SV (and those 
measures would not be repeated again until follow up). The reason for doing a prescreen visit is those two 
measures will often disqualify someone from the study so it greatly improves our work flow and scheduling 
abilities to schedule this as a separate visit since the PSV can be done in the afternoon whereas the SV is a 
fasting visit and has to be done in the morning. Following the screening visit, the study coordinator will check 
the lab results to determine if the participant is still eligible based on blood work (25(OH)D between 18 and <30 
ng/mL, liver and serum calcium within normal limits, and eGFR≥45). If the participant is eligible, they will be 
notified and scheduled for a baseline visit. If the participant is not eligible, they will be notified and given a copy 
of their screening blood work (if it was completed). 
 
At the first baseline visit 1 (BV1), lower extremity muscle power (Nottingham Power Rig) and strength 
(Biodex dynamometer), physical performance (the expanded SPPB, timed up and go (TUG), and 20-m walk 
speed), grip strength, postural sway (AMTI AccuSway force platform), timed stair climb, and gait velocity 
(GAITRite mat) will be measured. The CHAMPS Physical Activity Questionnaire for Older Adults,17 to estimate 
physical activity in a typical week over the past 4 weeks, and an arthritis and joint pain questionnaire will be 
administered. At the completion of BV1, the study coordinator will check to see if the participant has been 
selected to be in the muscle biopsy substudy and tell the participant whether or not he/she was selected to be 
in the muscle biopsy substudy. Participants selected for the muscle biopsy substudy will be given oral and 
written instructions to prepare for the muscle biopsy (not to take aspirin, ibuprofen, naproxen, or other 
medications that may affect bleeding, platelets, or bruising for 5 days before the procedure and to avoid 
strenuous physical activity for 36 hours before the procedure). Participants will be scheduled for their BV2 visit 
approximately one week later. 
 
 
Baseline visit 2 (BV2) will be conducted in the morning after an 8-hour fast in which participants will have 
blood drawn for analyses of serum 25(OH)D and 1,25(OH)D, plasma PTH, bioenergetic profiling (muscle 
biopsy subsample only), and blood for storage. For participants randomized to the muscle biopsy subsample, a 
muscle biopsy of the vastus lateralis (quadriceps) will also be taken. After vitals are collected and the blood 
draw (and muscle biopsy for those in the substudy) completed, participants will be given a snack. Participants 
in the muscle biopsy substudy will be given the option to continue with the BV2 visit following the muscle 
biopsy or to come back the following week to complete the BV2 questionnaires and be randomized in a 
separate visit. Then the Mobility Assessment Tool for Disability – short form (MAT-sf) to assess difficulty 
performing daily tasks, Digit Symbol Substitution Test (DSST) to assess attention and psychomotor speed, the 
Center for Epidemiologic Studies Depression Scale (CES-D) to measure mood and depression, and a sun 
exposure questionnaire (a modified version of the NHANES Dermatology Questionnaire) to estimate sun 
exposure and protection habits will be administered. At the conclusion of BV2, the study coordinator will check 
to see if the participant is randomized to receive either vitamin D3 or placebo. The study coordinator will 
provide the participant with a 4-month supply of vitamin D3 or placebo pills and monthly fall calendars to 
complete and provide oral and written instructions for taking the study supplement and completing the monthly 
fall calendars. Participants in the muscle biopsy substudy who do not elect to return the following week to 
complete the BV2 visit will receive a phone call 2-4 days following the procedure to check that they are not 
experiencing any adverse effects that may be related to the procedure (pain/soreness, signs of infection such 
as fever, excessive bleeding, or limitations in activity). The study physician (Dr. Demons) will be notified of any 
adverse effects related to the muscle biopsy procedure and any necessary action taken (e.g., follow-up visit 
scheduled with the study physician). 
 
Monthly phone calls: Participants will be called once a month throughout the study to ensure they are 
taking and tolerating the supplements and completing the monthly fall calendars. The study coordinator will ask 
about any falls or adverse events that may have occurred.  
 
The first 4-month follow-up visit (4FV1) will occur during week 16 of the intervention. After an 8-hour 
overnight fast, participants will have their vitals taken and blood drawn for analyses of serum 25(OH)D and 
1,25(OH)D, plasma PTH, metabolic panel, and storage followed by a snack. Lower extremity muscle power 
and strength, physical performance, grip strength, postural sway, stair climb, and gait velocity will be 
measured. The CHAMPS Physical Activity Questionnaire, MAT-sf, arthritis and joint pain questionnaire, and 
sun exposure questionnaire will be administered. Any changes to medical history and current medications and 

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dietary supplements will be reviewed. The first four months of fall calendars will be collected and reviewed. 
Participants will return the study supplement bottle with any remaining pills and an 8-month supply of vitamin 
D3 or placebo pills will be provided to them to last through the end of the study. Instructions for taking the study 
supplement and completing the monthly fall calendars will be reviewed. Participants in the muscle biopsy 
substudy will be scheduled for their follow-up muscle biopsy and given oral and written instructions to prepare 
for the procedure. Following the 4-month follow-up visit, the study coordinator will check the lab results to 
determine if the 25(OH)D concentrations are at or above 30 ng/mL in participants randomized to receive 
vitamin D3. Participants randomized to vitamin D3 who have not achieved 25(OH)D concentrations ≥30 ng/mL 
at the 4-month follow-up visit will be instructed to take 2 pills/d (4000 IU) for the remainder of the study. The 
study coordinator will also check to ensure that serum calcium levels are ≤10.6 mg/dL. If a participant’s serum 
calcium is >10.6 mg/dL at 4FV1, the protocol to assess whether the participant is hypercalcemic outlined in the 
Human Subjects Protection section (p. 10) will be followed. 
 
The second 4-month follow-up visit (4FV2) will occur 3-5 days after the 4FV1 visit in the morning for 
participants in the muscle biopsy substudy who had a viable muscle biopsy sample at BV2 only. After an 8-
hour overnight fast, participants will undergo a muscle biopsy of the vastus lateralis followed by a snack. 
Participants will receive a phone call 2-4 days following the procedure to check that they are not experiencing 
any adverse effects that may be related to the procedure. The study physician (Dr. Demons) will be notified of 
any adverse effects related to the muscle biopsy procedure and any necessary action taken. 
 
The 12-month follow-up visit (12FV) will occur during week 52 of the intervention. After an 8-hour overnight 
fast, participants will have their vitals taken and blood drawn for analyses of serum 25(OH)D and 1,25(OH)D, 
plasma PTH, metabolic panel, and storage followed by a snack. Lower extremity muscle power and strength, 
physical performance, grip strength, postural sway, stair climb, and gait velocity will be measured. The 
CHAMPS Physical Activity Questionnaire, MAT-sf, arthritis and joint pain questionnaire, DSST, CES-D, the 
MoCA, and sun exposure questionnaire will be administered. Medical history and current medications and 
dietary supplements will be reviewed. Monthly fall calendars will be collected and reviewed. Participants will 
return the study supplement bottles with any remaining pills. Participants will then be told of their randomization 
assignment. 
Muscle biopsies: Muscle will be obtained in a stratified sample of participants from the vastus lateralis 
using the percutaneous needle biopsy technique.18 The vastus lateralis is a good representative muscle for the 
in vitro study of skeletal muscle in older persons.19 By conducting an average of 3-4 passes (some may require 
additional passes depending on body composition) from a single incision, tissue samples of an average of 
500mg (more or less depending on body composition) are obtained, which is sufficient for the mechanistic 
analyses proposed and any remaining tissue will be frozen and stored for future use. Up to 40 participants may 
be approached for a baseline muscle biopsy to ensure that at least 32 participants complete the 4-month 
muscle biopsy and allow for biopsy samples that yield insufficient muscle for analyses. Once the specimen is 
obtained, visible blood and connective tissue is removed, and portions for specific assays are partitioned. A 
portion for histochemical analysis (≥20 mg) will be oriented such that the fibers run longitudinally, mounted in a 
histology tray in embedding medium (OCT compound, Miles Laboratory, Naperville, IL), and frozen in 
isopentane cooled to its freezing point with liquid nitrogen. Another portion to be used for RNA extraction (≥50 
mg) will be snap frozen in liquid nitrogen (within 30 seconds of the biopsy) and stored at -80°C until analyses. 
A portion of muscle tissue will be homogenized for extraction of mitochondria (≥50 mg). 
Vitamin D intervention 
Participants will be randomized to either 2000 IU/d of vitamin D3 or placebo for 12 months. The vitamin D3 
supplements and matching placebo pills will be manufactured by Tishcon Corp (Salisbury, MD). The proposed 
dose is expected to increase 25(OH)D concentrations by ~14 ng/mL.20,21 Previous vitamin D supplement 
dosing studies have observed a plateau in the increase in 25(OH)D concentrations in 6 to 8 weeks.20,22,23 In a 
recent dose-response study, a vitamin D3 dose of 1600 IU/d increased 25(OH)D concentrations from <20 
ng/mL to ≥30 ng/mL over 1 year in 97.5% of participants.24 However, we will measure serum 25(OH)D at the 4-
month visit and increase the vitamin D3 dose to 4,000 IU/d in participants in the intervention group with 
25(OH)D concentrations <30 ng/mL (for every participant in the intervention group that has to increase their 
vitamin D3 supplement dose to 2 pills/d after the 4-month follow-up visit, a random participant from the placebo 
group will also be instructed to take 2 placebo pills/d). Supplements will be dispensed at baseline and at the 4-
month visit. Participants will be instructed to maintain their regular dietary intake and typical physical activity 
level and instructed to refrain from starting any other dietary supplements or changing the frequency/dose of 
any supplements they are currently using during the study. A brown bag review will be done at each visit of all 

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dietary supplements participants are currently taking; and the brand and type, frequency, and dose of vitamin 
D and calcium will be recorded on the medication inventory. Supplement adherence will be monitored monthly 
via phone calls and the remaining supplement pills counted at the 4- and 12-month visits. Since the half-life of 
25(OH)D is ~2-3 weeks,27 occasional missed pills should have a minimal effect on 25(OH)D concentrations. 
The neuromuscular function assessments, mechanistic studies of the muscle biopsy sample, and assessments 
of 25(OH)D and 1,25(OH)2D will be conducted by study staff and investigators blinded to the vitamin D 
intervention assignment. 
Outcome measures 
All examiners are 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. Study outcomes (i.e., lower extremity muscle power and 
strength, expanded short physical performance battery, timed up and go, 20-m walk, stair climbing, grip 
strength, postural sway, gait velocity, and the mechanistic studies described below for the muscle biopsy 
substudy) will be assessed by blinded staff. 
Lower extremity muscle power will be measured using the Nottingham Power Rig, a safe, convenient 
method for assessing power output from the lower limb which has been used reliably in older adults.28 
Participants will sit in a chair and unilaterally depress a foot lever attached to a flywheel as hard and as fast as 
they can. Power output, derived from the acceleration of the flywheel from 5 trials on each leg at maximal 
effort, will be recorded in Watts. Participants that have had a unilateral hip or knee replacement should not 
have that side tested. Participants who have had eye surgery (e.g., cataract surgery) within the past month 
should not be tested. For the primary analyses, the ratio of the overall maximum leg power (right or left leg at 
baseline) to body weight in kg will be used and the maximum leg power from the same leg used at 4- and 12-
month follow-up. For analyses correlating muscle composition and physiology from the muscle biopsy 
substudy with leg power, the ratio of maximum leg power on the leg biopsied to body weight in kg will be used 
in analysis. 
 
Lower extremity muscle strength will be measured using an isokinetic dynamometer (Biodex) at one 
speed (60°/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°. Participants will be asked to extend the knee 
and push as hard as possible against the resistance pad. Strength is expressed as peak torque in Newton-
meters (Nm). Two trials will be done consisting of 4 repetitions each. Participants who have a history of brain 
aneurysm, cerebral bleeding in the past 6 months, or blood pressure >199/99 mmHg should not be tested. 
Participants that have had a unilateral knee replacement should not have that side tested. Participants who 
have had eye surgery (e.g., cataract surgery) within the past month should 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 4- and 12-month follow-up. For analyses 
correlating muscle composition and physiology from the muscle biopsy substudy with leg strength, the 
maximum leg strength on the leg biopsied will be used in analysis. 
Physical performance will be assessed using the expanded Short Physical Performance Battery (SPPB). 
The expanded SPPB consists of 5 repeated chair stands, standing balance (semi- and full-tandem stands and 
a single leg stand for 30 seconds), a 4-m walk to assess usual gait speed, and a narrow 4-m walk test of 
balance (walking at usual pace within lines of tape spaced 20 cm apart).29 Scores for the traditional 0-12 point 
SPPB can also be obtained from these tests. We will also assess physical performance using the Timed Up 
and Go (TUG). TUG measures the time a person takes to stand up from a standard chair, walk 3 m, turn, walk 
back to the chair, and sit down again.30 A practice trial is given, followed by a timed trial. Usual and fast walking 
speed over 20 m will be assessed to determine the functional reserve that exists in walking speed and to 
provide a substantive challenge over the usual pace 6-m walk used in the expanded SPPB. Stair climbing time 
will be assessed by measuring the time to climb up 4 steps on a 4-step staircase (test will be performed 2 
times). The faster of the 2 trials will be used in analysis. 
 
Grip strength will be measured twice in each hand to the nearest 2 kg using an isometric Hydraulic Hand 
Dynamometer (Jamar, Bolingbrook, IL) and the mean value from the stronger hand at baseline will be used in 
analysis and the mean value from the same hand used at 4- and 12-month follow-up. Participants will be 
excluded from performing the test if they report hand-pain or recent hand or wrist surgery.   

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Postural sway during quiet stance will be assessed from Center-of-Pressure (COP) trajectory data 
collected at 100 Hz using an Advanced Mechanical Technology Incorporated (AMTI) AccuSway biomechanics 
force platform. Participants will be barefoot in an upright stance with arms relaxed comfortably at their sides, 
feet abducted 10 degrees, and heels separated medio-laterally by 6 cm.31 COP data will be collected in a 
series of 10 30-sec trials standing on the force plate alone (closed trials) followed by 5 30-sec trials standing on 
the force plate with a 3 inch Airex foam pad (foam trials) placed on top. Four posturographic parameters 
(maximum anteroposterior (AP) and mediolateral (ML) displacement, average sway velocity, and 95% 
confidence ellipse) calculated by the AMTI Balance Clinic software will be used to quantify postural sway and 
the average of the closed and foam trials used in analysis.31 Studies have linked indicators of ML stability32-36 
or AP stability37 to clinical measures of balance performance and/or fall risk.  
Gait velocity will be measured using a 4.88 m long instrumented carpet (GAITRite, CIR Systems Inc., 
Clifton, NJ). The carpet is composed of 18,432 force sensing sensors arranged in a 48 wide x 384 long grid. 
Data are sampled at either 60, 120, or 240 Hz. The GAITRite is interfaced with a laptop computer that runs 
software to calculate variables related to gait performance. The GAITRite system is a valid instrument for 
determining gait velocity and other spatiotemporal parameters. In the testing procedures, participants walk 
across the carpet, beginning 2 m in front of the carpet and walking 2 m past the end of the carpet to allow for 
acceleration and deceleration. Participants make multiple passes over the carpet at either the preferred (4 
trials) or fast (4 trials) velocity. At the preferred velocity, participants are told to walk at “at a pace that feels 
comfortable to you (as if you were walking to the mailbox or window shopping at the mall).” At the fast velocity 
the instructions are to walk “as quickly as you can without running.” The software calculates individual trial and 
average data on gait velocity and spatiotemporal gait characteristics and these data can be exported for further 
analysis. Summary data on velocity (m/sec; averaged over the 4 usual and 4 fast trials) and the average of the 
left and right leg for double support (percent of cycle), single support (percent of cycle), stance (percent of 
cycle), swing (percent of cycle), stride length (cm), and base width (cm) for both the usual and fast trials will be 
used in analysis.  
Falls will be defined as an event whereby an individual unexpectedly comes to rest on the ground or 
another lower level. Participants will be provided monthly fall calendars, considered the “gold standard”,38 and 
asked to mark any falls that occur on the calendar. Completion of monthly fall calendars will be monitored via 
monthly phone calls and collected at the 4- and 12-month visits. Total number of falls over the 12-month follow-
up will be used in analysis. 
Single fiber-type composition and fiber size will be examined in muscle biopsies using the ATPase, pH 
9.4 and 4.6 technique, combined with laminin immunostaining in 10 µm muscle sections and the absolute and 
relative number of type I, IIA, IIX fiber subtypes and their cross-sectional area quantified. The relative number 
of fiber type and their cross-sectional area at baseline and 4-month follow-up will be used in analysis.   
Single type I and type II muscle fiber contraction force, shortening velocity, and power will be 
examined in 10-15 fibers per muscle biopsy sample. For single muscle fiber preparation, physiologic tests, and 
experimental procedures, we will follow described techniques.39,40 Briefly, a slack test will be used to determine 
the unloaded shortening velocity (Vo). The force/velocity relationship will be determined by performing a series 
of isotonic contractions of the muscle fiber.41 The muscle fiber will be placed in activating solution (pCa 4.5) 
and, after it reaches peak force, subjected to three isotonic steps varying from 3-80% of Po. After the final step, 
the fiber is rapidly (<1ms) slackened by 20% of its length. This zeroes the force transducer, providing a 
baseline for force measurement. Shortening velocity and force will be assessed over the final half of each step, 
and force averaged over the last half of each step. Velocity will be calculated as the slope of the position 
recorded over the same time period. A total of 5-6 isotonic contraction series will be performed, generating 15-
18 pairs of velocity and relative force measurements. Data will be fit to the Hill equation using an iterative, 
nonlinear curve-fitting procedure to draw the force/power relationship. All forces will be normalized to the fiber’s 
cross sectional area. At the end of each functional experiment, the single-fiber segment will be used to 
determine myosin heavy chain (MHC) composition using silver staining.41,42 Fibers will be excluded from 
analysis if force declines more than 10% or if fibers break or show partial myofibrillar tearing during the 
experimental protocol. Absolute fiber force (slk max mN), normalized fiber force to cross-sectional area (slk pk 
kN/m2), unloaded shortening velocity (Vo, FL/s), the velocity extrapolated to a force of zero (FV Vmax, FL/s), 
power (uN*FL/s), and normalized power (W/liter) by fiber type (I or II) at baseline and 4-month follow-up will be 
used in analysis. 

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Type I and type II denervated myofibers will be identified using ATPase pH 9 plus NCAM 
immunostaining analysis. The whole muscle biopsy will be cryosectioned and 10-12 slices will be mounted per 
slide. Alternate slides will be stained for NCAM and positive fibers and their expression pattern (perimeter or 
whole cross section) will be reported as a percent of the total number of fibers. Additionally, fiber grouping (the 
area occupied by at least two contiguous fibers expressing a predominantly similar myosin heavy chain isoform 
– type I or II, fully surrounded by fibers of the same predominant myosin heavy chain), a classical evidence of 
a neurogenic process, will be examined. NCAM+ number and density (NCAM+ number per cross sectional 
area) and fiber grouping (%) by fiber type (I or II) at baseline and 4-month follow-up will be used in analysis. 
Regulation of Runx1 and its dependent genes will be assessed by RNA extraction using the TRIzol 
method (Invitrogen). Runx1 and its dependent genes will be measured by qRT-PCR and normalized to 
GAPDH expression. We will use the cycle threshold (CT), defined as the number of cycles required for the 
fluorescent signal to cross the threshold, as the parameter to calculate 2-CT, which is needed to compare the 
gene expression in two different samples. Expression of Runx1, Chrn, Scn5a, Myh3, Myog, NCAM1 at 
baseline and 4-month follow-up will be used in analysis. 
Satellite cell differentiation will be assessed in serial muscle cross sections immunostained for 
Pax7/NCAM satellite cells (SCs). SC number and density (SC per cross sectional area) by fiber type (I or II) at 
baseline and 4-month follow-up will be used in analysis. 
Vitamin D Receptor (VDR) expression will be measured by Western blots using the C20 antibody raised 
against a peptide mapping at the C terminus of VDR and normalized to actin used as a loading control. VDR 
concentration at baseline and 4-month follow-up will be used in analysis. 
Exploratory outcomes (Wake Forest Pepper Center Pilot; Anthony Molina, PI) 
Respirometric profiling will be used to assess bioenergetic capacity, respiratory control and electron 
transport chain function. This will be performed by examining the oxygen consumption profile of blood cells and 
mitochondrial fractions extracted from muscle tissue. Specific outcomes of mitochondria function include 
respiratory control ratio (RCR), state 3 (maximal oxygen consumption rate), and state 4o (oxygen consumption 
upon inhibition of ATP synthase).  
Respirometry of isolated mitochondria will be performed in order to examine intrinsic mitochondrial function 
(type 1). Purified mitochondria with intact electron transport chain activity will be obtained from muscle tissue. 5 
ug of mitochondria will be loaded into each well of V7 Seahorse plates for analysis using an XF-24-3 system 
(Seahorse Bioscience, Billerica MA). Specific measures obtained include: baseline respiration (state 2), 
maximal respiration (state 3), maximal uncoupled respiration (state 3u), and proton leak (state 4). 
Mitochondrial mass and biogenesis will be determined by western blot analysis. Changes in mitochondrial 
mass will be assessed using VDAC/porin and COXIV expression. PGC1a, TFAM, SIRT1 and SIRT3, 
regulators of mitochondrial biogenesis, will also be measured on the same immunoblot. All primary antibodies 
will be obtained from Abcam (Cambridge, MA). Quantification of each immunoreactive product will be 
performed by densitometry using Kodak (Rochester, NY) imaging system.   
Blood-based bioenergetic profiling. PBMC’s, monocytes, lymphocytes, and platelets will be separated from 
whole blood and assessed for basal respiration, maximal respiration, ATP linked respiration, spare respiratory 
capacity, proton leak, and non-mitochondrial respiration as described above. 
Covariates 
Vitamin D status: Fasting blood samples will be collected in the morning by trained and certified 
phlebotomists at baseline and 4-month and 12-month follow-up. Blood samples will be sent to LabCorp for 
assessment of serum 25(OH)D for screening purposes and at 4-month follow-up to assess 25(OH)D response 
to vitamin D supplementation and for assessment of serum calcium at each visit. After processing, 
approximately 4 ml of the blood from each visit will be aliquotted and stored at -80ºC until it is shipped to the 
Clinical and Analytical Lab (CACL) at the Human Nutrition Research Center on Aging at Tufts University where 
the samples will be stored at -80ºC until analysis. Serum total (D2 and D3) 25(OH)D will be assessed in 
duplicate using LC/MS/MS (Waters Acquity UPLC with TQD triple quadrupole mass spectrometer; CV: 6%) 
along with the 25(OH)D standard reference material (SRM 972) developed by the National Institute of 
Standards and Technology and the National Institutes of Health Office of Dietary Supplements for assay 
calibration.45 Serum 1,25(OH)2D will also be assessed in duplicate using the 5500 QTRAP LC/MS/MS System 
(AB Sciex LLC) with Electrospray after immunoaffinity extraction, 4-phenyl-1,2,4-triazole-3,5-dione (PTAD) 

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derivatization, and methylamine adduction (intra- and interassay CVs of 5% and 8.5%, respectively). Repeat 
analyses will be conducted when duplicate samples differ by 10% or more. Storage: Approximately 10 ml of 
blood (serum and plasma) from each visit will be stored at -80ºC in the Pepper Tissue Repository (IRB# 1219) 
for storage. Any person wishing to use this blood will obtain IRB approval first.  
Season: The season in which the participant is screened for eligibility will be obtained to examine if the 
season of enrollment affects the outcomes due to seasonal effects on endogenous vitamin D synthesis and 
25(OH)D concentrations.  
Supplement adherence: Although we will attempt to enhance adherence by monthly calls to participants, 
there may be inter-individual variability in adherence to supplementation. At the 4 and 12-month study visits, 
participants will be asked to return any unused supplements for a pill count to determine participant adherence. 
Our primary analyses will be an intent-to-treat analysis of the effects of the vitamin D intervention, but 
secondary analyses will include adherence as a covariate. 
Calcium and vitamin D intake: Calcium and vitamin D intake from diet and supplements will be assessed at 
baseline using a short calcium and vitamin D intake questionnaire.  
Cognitive function will be assessed during screening using the Montreal Cognitive Assessment (MoCA), 
participants must score ≥18 to be eligible. We will also assess psychomotor speed, attention, and working 
memory using the Digit Symbol Substitution Test (DSST). 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). The cognitive function tests will be audio recorded by the 
assessor using a hand held digital recorder. The recordings will be used to properly score the tests as well as 
be available for quality control testing. The recordings will be deleted once the study is over and the data that 
was entered has been verified. 
Mood will be assessed using the 20-item Center for Epidemiologic Studies Depression Scale (CES-D). 
Demographic characteristics including age, gender, race, education level, and socio-economic status will 
be recorded and used as covariates if necessary.  
Body mass index (BMI) will be calculated from measured height and weight.  
Physical activity will be measured using the CHAMPS Physical Activity Questionnaire for Older Adults17 at 
baseline, 4- and 12-month follow-up. In addition to being a strong correlate of the neuromuscular function 
measures, more physically active participants will likely have increased sun exposure, and therefore, may have 
higher endogenous vitamin D production.  
Activities of daily living will be assessed using the Mobility Assessment Tool – Short Form (MAT-sf) at 
baseline, 4- and 12-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.  
Sun exposure will be assessed at baseline, 4- and 12-month follow-up using a modified version of the 
NHANES Dermatology Questionnaire that includes questions on sun exposure and sun protection habits.  
Medical information regarding co-morbidities will be ascertained at the screening visit and reviewed at the 
4- and 12-month follow-up visits. Arthritis and joint pain will be assessed by self-report on a questionnaire. We 
will record medication and supplement use at screening and 4- and 12-month follow-up. Participants will also 
be asked to bring in medications and dietary supplements for a "brown bag" review. The medication form will 
specifically ask about the use of vitamin D-containing supplements, anti-inflammatory agents, protein 
supplements, and sex steroids or corticosteroids. 
Statistical Power  
We anticipate that, based on the vitamin D3 supplement dose of 2000 IU/d, 25(OH)D concentrations will 
increase by an average of 14 ng/mL in the intervention group at 12-month follow-up. In preliminary data from 
17 older individuals with vitamin D concentrations between 20 and 40 ng/mL, we found that a 1 ng/mL 
difference in 25(OH)D concentration was associated with a 0.0233 watts/kg of body weight difference in lower 
extremity muscle power for men and women combined. If this cross-sectional relationship holds for the 
intervention study, we would expect approximately a 0.33 watts/kg difference between the two groups at 
follow-up (0.0233 x 14 = 0.33). However, due to the uncertainty of extending cross-sectional data to 
longitudinal follow-up and the potential for non-adherence, we have based the power on a difference in means 

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Table 3. Differences detected with 80% power for secondary outcomes  
Outcome measure 
SD* 
Detectable 
difference 
Short Physical Performance Battery score30 
2.05 
0.92 
Timed Up and Go, sec82 
5.40 
2.43 
20-m Gait Speed, m/sec31 
0.15 
0.07 
Lower extremity muscle strength, Nm31 
21.0 
9.5 
Postural Sway, mm122 
     Mediolateral (ML) displacement 
     Anteroposterior (AP) displacement 
 
2.7 
3.9 
 
1.2 
1.8 
*SD calculated assuming a correlation of 0.43 between baseline and follow-
up measures. 
of a 10% relative change between the two groups; a magnitude that resulted in a clinically meaningful increase 
on the SPPB in a previous intervention trial.47 The mean (SD) of lower extremity muscle power was 1.74 
(0.382) watts/kg in our preliminary data; a 10% difference (0.174 watts/kg) corresponds to an effect size of 
0.45. A sample size of 125 participants randomized in a 1:1 ratio stratified by gender and race will have >80% 
power to detect this difference using analysis of covariance (ANCOVA), adjusting for the baseline measure, 
based on a baseline mean of 1.7 watts/kg (SD=0.444) and a correlation between baseline and 12-month 
follow-up of 0.708 with a 0.05 two-sided significance level. Allowing for a 10% dropout rate, we will inflate the 
sample size to 136. We will also have sufficient power to detect a difference of 0.45 SD or greater in our 
secondary outcomes (Table 3).  
For the muscle biopsy subsample, we 
have powered on a difference between the 
two groups of a relative increase in type II 
fibers of 25% or more. In our preliminary data 
in older adults with 25(OH)D concentrations 
<25 ng/mL treated with 2000 IU/d vitamin D3 
plus 1200 mg/d calcium (n=4) or calcium only 
(n=2), a 1 ng/mL increase in 25(OH)D 
concentration was associated with a 2.35% 
relative increase in type II fibers over 4 
months. Assuming that 25(OH)D 
concentrations will increase by an average of 
14 ng/mL in the intervention group, we 
expect to see a relative increase in type II fibers of approximately 33%. To allow for the possibility that there 
may be some increase in the control group due to “drop in” by starting vitamin D supplements or changes in 
physical activity, a 25% difference between the two groups in the relative increase in type II fibers was 
selected. Two previously published vitamin D supplementation studies observed increases in type II fibers of 
31-34%48 and 202%.49 Thus, the detectable 25% relative increase is in line with independent observations. A 
sample size of 16 in each group will have 80% power to detect a difference of 25% assuming that the common 
standard deviation is 23.7 (based on preliminary data of n=17 participants in a behavioral intervention with data 
on change in type II muscle fibers) using a two group t-test with a 0.05 two-sided significance level. Allowing 
for 10% drop out, we will inflate the sample size to 18 per group, for a total of 36 participants. However, up to 
40 participants may be approached for a baseline muscle biopsy to allow for biopsy samples that yield 
insufficient muscle for analyses. 
Statistical Analyses 
 
We will examine the distributions of all continuous outcomes of interest, and will transform them to 
approximate normality if the outcomes show a strong departure from normality as evidenced by examining Q-Q 
plots and performing the Shapiro-Wilk test. Outliers will be examined on the chosen scale (original or 
transformed) and may be set to missing if they are 3 or more interquartile ranges above the 75th percentile or 
below the 25th percentile. Plausible biologic values may be retained even if they are outliers. Implausible 
biologic values may be set to missing based on a review of health-related events (e.g., AEs related to pain, 
fractures, surgery) in the interval surrounding the collection of the measurements. 
 
The primary outcome for this trial will be the 12-month measure of muscle power. For the muscle biopsy 
substudy, the primary outcome will be the change in type II muscle fibers over 4-months. Our primary statistical 
analyses will proceed on the “intent-to-treat” principle in which data from all randomized participants are used 
to test the primary hypotheses that compared to placebo, vitamin D3 supplementation will result in increases in 
lower extremity muscle power and strength, and improvements in physical performance (expanded SPPB, 
TUG, and 20-m walk speed) and balance. Even if participants wish to discontinue the supplements, we will still 
attempt to follow them for outcomes in order to carry out intent-to-treat analysis at 12 months. Characteristics 
of the participants missing the 12-month follow-up outcomes will be compared to participants without missing 
data using chi-square tests and t-tests to determine if there are any differences with regard to intervention 
group, demographic characteristics, and baseline neuromuscular function outcomes. If a particular group 
exhibits markedly higher drop-out rates, we will attempt to identify baseline covariates that predict attrition. If 
such covariates can be identified, the analyses of outcome measures may need to be stratified according to 
these comparisons to decrease bias. We will use a mixed model with an unstructured covariance matrix to 
model after-treatment neuromuscular function outcomes between the two groups by time, with adjustment for 

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the baseline neuromuscular function measure, age, gender, and race, using data from baseline, 4, and 12 
months, and estimating the baseline to 12 month change using linear contrasts. In secondary analyses, we will 
fit linear contrasts to compare whether the change between the two groups at 4 months differs from the change 
at 12 months. These analyses will be adjusted for age, gender, race, and season, as we anticipate there will be 
an effect of season on the change from baseline and 12 months. If the analyses of the characteristics of those 
missing the 12-month outcome data indicate that the missing data are informative, we will also consider using 
the 4-month outcome data to impute the 12-month outcome, using the trajectory models to predict the 12-
month outcome. 
 
We will examine factors that may influence the measured responses to the intervention. We anticipate that 
not all individuals will respond to the vitamin D3 dose in a similar manner due to genetic differences50-52 as well 
as differences in BMI,53,54 health status, and adherence.3 First, individuals in the vitamin D intervention group 
who have low adherence (<80%) will be excluded from the analysis. We will also investigate treatment effects 
by attained 25(OH)D concentrations including only those in the intervention group whose 12-month 25(OH)D 
concentrations are ≥30 ng/mL. We will also fit models that include BMI, health status, and other variables to 
determine if they account for any of the variation in response to the intervention on changes in the outcomes. 
For our exploratory outcome of falls, we will use a generalized estimating equation (GEE) approach to model 
repeated measures, assuming a Poisson distribution, to compare the rate of falls in each group with 
adjustment for age, gender, and race.55 Differences in, or reasons for, attrition rates between study groups may 
complicate the analysis of the primary outcomes. Therefore, the attrition rates will also be compared and, if a 
particular group exhibits markedly higher drop-out rates, we will attempt to identify baseline covariates that 
predict attrition. If such covariates can be identified, the analyses of outcome measures may need to be 
stratified according to these comparisons to decrease bias.  
 
For the muscle biopsy subsample (n=16 per group), primary analyses will proceed on the “intent-to-treat” 
principle in which data from all randomized participants are used in analyses. All participants who complete the 
4-month muscle biopsy and on whom the muscle fiber testing can be completed will be included in the analytic 
sample. Even if participants wish to discontinue the supplements, we will still attempt to follow them for 
outcomes in order to do the intention-to-treat analysis at 4 months. Characteristics of the participants missing 
the 4-month follow-up outcomes will be compared to participants without missing data using chi-square tests 
and t-tests to determine if there are any differences with regard to intervention group, demographic 
characteristics, baseline physical performance, and baseline muscle fiber testing. Our primary statistical 
analyses will focus on testing the primary hypotheses that, compared to placebo, vitamin D supplementation 
will result in changes in skeletal muscle composition and physiology as quantified by: fiber-type composition 
and size; fiber force, shortening velocity, and power; NCAM+ myofibers; Runx1, Chrn, Scn5a, Myh3, Myog, 
and NCAM1 expression; SC number; VDR expression; and bioenergetic capacity. For our primary outcome of 
percent type II fibers we will use analysis of covariance (ANCOVA) to model differences in percent of type II 
fibers between the two groups, with adjustment for the baseline measure, age, gender, and race. Analysis of 
other continuous measures will also use the ANCOVA model to compare the groups adjusted for age, gender, 
and race. In secondary analyses, we will examine if the percent of participants who have a relative increase in 
type II fibers of 25% or more from baseline differs by intervention group using Fisher’s Exact Test, and in 
logistic regression models adjusted for age, gender, and race. Furthermore, we will explore if the relationship 
between vitamin D supplementation and neuromuscular function is mediated by change in muscle composition 
and physiology by including the variables described above as covariates in the ANCOVA model described 
above. If the relationship between vitamin D supplementation and power decreases by 10% or more, it will be 
considered evidence of partial mediation. 
Human Subjects Protection 
Subject Recruitment Methods 
     We will recruit these individuals using community-based recruitment strategies including newspaper ads 
and mass mailings. We will also advertise in the VITAL newsletter (BG99-559) and participate in community 
outreach events.  
Informed Consent 
 
Written informed consent will be obtained from each subject. The informed consent process will follow the 
procedures of the WFSM 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 

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consent form and ask questions. The form is 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 provided with a 
structured checklist for this purpose. 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 Risks 
1) Neuromuscular function.  There is a small risk of injury during the muscle strength and power testing, 
stair climb, postural sway and gait velocity 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) Blood Draw. Participants may experience temporary pain, bruising, bleeding and a small  
risk of infection or fainting or dizziness during the blood sample collection process. Only trained staff will 
be responsible for the collection of blood samples.  
3) Muscle biopsy. There may be some temporary discomfort and bleeding from the muscle biopsy. 
However, there is typically no discomfort felt during the procedure due to the use of lidocaine as a local 
anesthetic injected into the muscle. Temporary numbness of the skin near the sampling site can rarely 
occur. A small amount of bruising can also occur. There is a slight risk of bleeding into the tissue and 
infection, but this procedure is done under sterile conditions to protect against this. There is also a risk 
of a vaso-vagal response during or after this procedure, but there are trained medical personnel 
performing the procedure and a fully stocked crash cart is available should it be needed. To date, Dr. 
Demons and Dr. Lyles have performed >600 muscle biopsies for aging-related research studies without 
a serious adverse event. 
4) The risks of the vitamin D supplement intervention are minimal, but may include hypervitaminosis D. 
Although the proposed vitamin D3 dose of 2000 IU/d is below the upper limit set by the Institute of 
Medicine (4000 IU/d), participants could exceed the upper limit if they begin consuming other vitamin D-
containing supplements. Participants will be advised not to begin any new dietary supplements during 
the trial and of the possible side effects of hypervitaminosis D (e.g., non-specific symptoms such as 
anorexia, weight loss, polyuria, and heart arrhythmias; as well as increased serum calcium which can 
lead to vascular and tissue calcification including kidney stones). Those with contraindications to 
vitamin D supplements will be excluded (e.g., kidney impairment (eGFR <45 mL/min/1.73), 
hypercalcemia, sarcoidosis, and history of kidney stones). Serum calcium will be assessed at screening 
and at 4- and 12-month follow-up. Participants with hypercalcemia (serum calcium >10.6 mg/dL) at 
their 4-month visit will have a repeat serum calcium drawn within 2 weeks, and, if confirmed, the study 
supplement will be discontinued; however, we will continue following participants with confirmed 
hypercalcemia for outcomes.  
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. The information collected has a low 
potential for abuse because the data do not address sensitive issues. Nevertheless, appropriate measures are 
taken to prevent unauthorized use of study information. Research records are kept in a locked room. Data 
access will be limited to study staff. Information linking IDs to individuals is kept on a secure, password-
protected server to which only authorized study personnel will have access. Data and records will be kept 
locked and secured, with any computer data password protected. A web-based data entry system will be 

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created for this study which will allow access only to study staff (user id and password required to 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 the research personnel, who are asked 
to sign a document agreeing to maintain the confidentiality of the information. No data files distributed for 
analysis will include personal information. No reference to any individual participant will appear in reports, 
presentations, or publications that may arise from the study. After the study is completed, the data will be 
stored with other completed research studies in a locked secure area.  
Data and Safety Monitoring 
 
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. A Data Safety 
Monitoring Board (DSMB) has been established by the NIH, with responsibility to monitor all aspects of the 
study, including those that require access to any blinded data. The operational plan and all members were 
approved by the NIH Program Scientist. The NIH-appointed DSMB responsibilities are to:  
– 
review the research protocol, informed consent documents and plans for data safety and 
monitoring;  
– 
advise the NIA on the readiness of the study staff to initiate recruitment; 
– 
evaluate the progress of the trial, including periodic assessments of data quality and timeliness, 
recruitment, accrual and retention, participant risk versus benefit, performance of the trial sites, and 
other factors that can affect study outcome; 
– 
consider factors external to the study when relevant information becomes available, such as 
scientific or therapeutic developments that may have an impact on the safety of the participants or 
the ethics of the trial; 
– 
review study performance, make recommendations and assist in the resolution of problems 
reported by the Principal Investigator; 
– 
protect the safety of the study participants; 
– 
report to NIA on the safety and  progress of the trial;  
– 
make recommendations to the NIA, the Principal Investigator, and, if required, to the Food and Drug 
Administration (FDA) concerning continuation, termination or other modifications of the trial based 
on the observed beneficial or adverse effects of the treatment under study; 
– 
ensure the confidentiality of the study data and the results of monitoring; and,  
– 
assist the NIA by commenting on any problems with study conduct, enrollment, sample size and/or 
data collection. 
 
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 Medical Director will meet quarterly, or as needed, to review all reported events. 
 
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 event, i.e., any serious injury or life-threatening event, will be reported immediately 
right after completing any and all actions that are necessary to protect the subject’s health and safety. 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. 
In addition, participant safety in all Wake Forest Pepper Center OAIC-supported studies is monitored by a 
Data and Safety Monitoring Committee (DSMC). It reviews study recruitment, drop-outs, protocol changes, 

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losses to follow-up, and adverse events every 6 months. The DSMC has authority to recommend changing or 
stopping the protocol. In the latter case, the PI will immediately act upon the recommendation according to 
institutional policy and in consultation with the Wake Forest Pepper Center OAIC Executive Committee and the 
NIH-appointed DSMB committee.   
Reporting of Unanticipated Problems, Adverse Events or Protocol Deviations 
Any unanticipated problems, serious 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 or muscle samples. 
Storage and disposal of biological material 
Blood and muscle samples will be stored at Wake Forest Baptist Medical Center for 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 Revco freezers located in a locked room. The Wake Forest Pepper Center Integrative Biology 
Core lab coordinator (Heather Gregory) and the Core Co-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). 
 
References 
 
(1)  Institute of Medicine, Committee to Review Dietary Reference Intakes for Vitamin D and Calcium, Food 
and Nutrition Board. Dietary reference intakes for calcium and vitamin D. Washington, D.C.: The 
National Academies Press; 2011. 
 
(2)  Bischoff-Ferrari HA, Giovannucci E, Willett WC, Dietrich T, Dawson-Hughes B. Estimation of optimal 
serum concentrations of 25-hydroxyvitamin D for multiple health outcomes. Am J Clin Nutr 
2006;84(1):18-28. 
 
(3)  Dawson-Hughes B, Mithal A, Bonjour JP, Boonen S, Burckhardt P, Fuleihan GE, Josse RG, Lips P, 
Morales-Torres J, Yoshimura N. IOF position statement: vitamin D recommendations for older adults. 
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