The Vitality, Independence, and Vigor in the Elderly 2 Study (VIVE2): Design and Methods

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Contemporary Clinical Trials 43 (2015) 164–171

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The Vitality, Independence, and Vigor in the Elderly 2 Study (VIVE2):Design and methods

Dylan R. Kirn a,⁎, Afsaneh Koochek b, Kieran F. Reid a, Åsa von Berens b, Thomas G. Travison f,g,m, Sara Folta d,Jennifer Sacheck d, Miriam Nelson d,e, Christine Liu a,i, Edward Phillips a,g,h, Anna Cristina Åberg j,k,Margaretha Nydahl l, Thomas Gustafsson c, Tommy Cederholm b, Roger A. Fielding a

a Nutrition, Exercise Physiology, and Sarcopenia Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA, United Statesb Clinical Nutrition and Metabolism, Department of Public Health and Caring Sciences, Uppsala University, Uppsala, Swedenc Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Swedend Friedman School of Nutrition Science and Policy, Tufts University, Boston, MA, United Statese Jonathan M. Tisch College of Citizenship and Public Service, Tufts University, Medford, MA, United Statesf Institute for Aging Research, Hebrew SeniorLife, Boston, MA, United Statesg Harvard Medical School, Boston, MA, United Statesh Spaulding Rehabilitation Hospital, Boston, MA, United Statesi Section of Geriatrics, Boston University School of Medicine, Boston, MA, United Statesj Geriatrics, Department of Public Health and Caring Sciences, Uppsala University, Uppsala, Swedenk School of Education, Health and Society, Dalarna University, Swedenl Department of Food, Nutrition and Dietetics, Uppsala University, Uppsala, Sweden.m Division of Gerontology, Beth Israel Deaconess Medical Center, Boston, MA, United States

⁎ Corresponding author at: Nutrition, Exercise PhysioloJean Mayer USDA Human Nutrition Research Center oWashington Street, Boston, MA, United States.

E-mail address: [email protected] (D.R. Kirn).

http://dx.doi.org/10.1016/j.cct.2015.06.0011551-7144/© 2015 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 19 March 2015Received in revised form 27 May 2015Accepted 1 June 2015Available online 2 June 2015

Keywords:Older adultsMobility-limitationsPhysical activityProteinVitamin DSupplementation

Background: Nutritional supplementation may potentiate the increase in skeletal muscle protein synthesisfollowing exercise in healthy older individuals. Whether exercise and nutrition act synergistically to producesustained changes in physical functioning and body composition has not been well studied, particularly inmobility-limited older adults.Methods: The VIVE2 study was a multi-center, randomized controlled trial, conducted in the United States andSweden. This studywas designed to compare the effects of a 6-month interventionwith a once daily, experimen-tal, 4 fl. oz. liquid nutritional supplement providing 150 kcal, whey protein (20 g), and vitaminD (800 IU) (NestléHealth Science, Vevey, Switzerland), to a low calorie placebo drink (30 kcal, non-nutritive; identical format)when combinedwith group-based exercise in 150 community-dwelling,mobility-limited older adults. All partic-ipants participated in a structured exercise program (3 sessions/week for 6 months), which included aerobic,strength, flexibility, and balance exercises.Results: The primary outcome was 6-month change in 400 mwalk performance (m/s) between supplement and

placebo groups. Secondary outcomes included 6 month change in: body composition, muscle cross-sectionalarea, leg strength, grip strength, stair climb time, quality of life, physical performance, mood/depressive symp-toms and nutritional status. These outcomes were selected based on their applicability to the health and well-being of older adults.Conclusions: The results of this study will further define the role of nutritional supplementation on physicalfunctioning and restoration of skeletal muscle mass in older adults. Additionally, these results will help refinethe current physical activity and nutritional recommendations for mobility-limited older adults.

© 2015 Elsevier Inc. All rights reserved.

gy, and Sarcopenia Laboratory,n Aging, Tufts University, 711

1. Introduction

Mobility limitations, such as the inability to rise from a chair orascend a flight of stairs, affect many older adults. Mobility-limitedolder adults experience higher rates of falls, institutionalization, andchronic disease [1]. As the worldwide population of persons 70 yearsand older continues to increase in the United States [2] as well as in

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Table 1Inclusion/exclusion criteria.

Inclusion criteriaMales and females age ≥70 yearsCommunity dwellingShort physical performance battery ≤9 [34]Willingness to be randomized and come to the laboratory for 6 monthsBody mass index ≤35Mini mental state examination ≥24 [35]Serum 25 (OH) D 22.5–60 nmol/lHaving obtained his/her informed consentAble to complete 400 M walk within 15 min

Exclusion criteriaCurrent health status

Acute or terminal illnessMyocardial infarction in previous 6 months, symptomatic coronary arterydisease, or congestive heart failureUpper or lower extremity fracture in previous 6 monthsUncontrolled hypertension (N150/90 mm Hg)Hemoglobin b10 g/dlEstimated GFR b30 ml/min/1.73 m2

Major surgery in the past 6 months (requiring general anesthesia)Other significant co-morbid disease that would impair ability to participate in anexercise-based intervention, e.g. renal failure on hemodialysis, severepsychiatric disorder (e.g. bipolar, schizophrenia)Wheelchair boundInability to communicate due to severe, uncorrectable hearing loss or speechdisorderSevere visual impairment (if it precludes completion of assessments and/orintervention)Severe progressive, degenerative neurologic diseaseSevere rheumatologic or orthopedic diseases, e.g., awaiting joint replacement,active inflammatory diseaseTerminal illness with life expectancy less than 12 months, as determined by aphysicianCancer requiring treatment in the past 3 years, except for non-melanoma skincancers or cancers that have clearly been cured or in the opinion of theinvestigator carry an excellent prognosis (e.g., stage 1 cervical cancer)Severe pulmonary disease, requiring either steroid pills or injections or the useof supplemental oxygenSevere cardiac disease, clinically significant aortic stenosis, history of cardiacarrest, use of a cardiac defibrillator, or uncontrolled angina

Nutritional exclusionsCurrent regular use of high protein oral supplements (N1 per week)Vitamin D supplements N800 IU/dayMilk protein allergyExcessive alcohol use (N14 drinks per week)

Physical activityParticipation in moderate intensity activity N20 min/week

BehavioralPatient who cannot be expected to comply with treatment as decided by thePrincipal InvestigatorConditions not specifically mentioned above may serve as criteria for exclusionat the discretion of the clinical site Principal Investigator and/or study physician

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Europe [3], there is a substantial need for interventions to address theinevitable increase in mobility limitations among this population.

Inadequate physical activity and low protein intake are two factorsthat contribute to the decline in muscle mass and function associatedwith aging (sarcopenia) [4–6]. Though several studies have shown apositive impact of physical activity in this population [7–9], studies in-vestigating protein supplementation in combination with exercisehave been mixed [10–17]. Several lines of evidence have suggestedthat nutritional supplementation (particularly supplements high in pro-tein) can potentiate the increase in skeletal muscle protein synthesisfollowing a single bout of exercise in both young individuals and inhealthy older adults [18–21], though the effects in mobility-limitedolder adults remain unclear. More recently, poor vitamin D status hasemerged as a potential contributor to poor muscle function amongolder adults [22]. Observational evidence suggests that individualswith deficient or insufficient vitamin D levels aremore functionally lim-ited compared to those who are sufficient [23]. Additionally, cross-sectional studies have shown that vitamin D status is highly correlatedwith lower-extremity strength and balance [24–27]. Though it hasbeen hypothesized that, vitamin D supplementation with and withoutexercise may decrease incidence of falls and fractures in the elderly,the results of these trials have been mixed [27–29]. However, bothcross-sectional and randomized controlled trialswith vitaminD supple-mentation that investigatemuscle function have had conflicting results,and therefore this relationship requires further investigation [30,31].

To date, no studies have been conducted to determinewhether syn-ergistic effects of exercise and nutrition can result in sustained increasesin physical functioning and fat free mass. Such investigations would beof particular relevance to older adults with demonstrated functionallimitations, who are at disproportionate risk for mobility disability.The Vitality, Independence, and Vigor in the Elderly 2 Study (VIVE2) de-scribed here was designed to compare the effects of an interventionwith an experimental liquid oral nutritional supplement providing150 kcal, high amounts of whey protein (20 g), and high amounts of vi-tamin D (800 IU) in a small volume (4 fl. oz., ca. 119 ml; (Nestlé HealthScience, Vevey, Switzerland)), to a low calorie placebo drink (ca. 30 kcal,noprotein, noVitaminD; identical format) on exercise training-inducedchanges in physical functioning in older adults with demonstratedmobility limitations and vitamin D insufficiency (≤60 nmol/l) acrosstwo international field centers.

1.1. Primary research outcome

The primary outcome of the VIVE2 study was 6 months change inwalking speed, as derived from the 400meterwalk test [32]. The designwas intended to facilitate comparison of the effects of a 6 months struc-tured physical activity program in combination with daily high protein,high vitamin D nutritional supplementation or placebo on changes inwalking speed and other outcomes, using data on 150 older adultswith limitations in mobility at baseline.

2. Methods

2.1. Overview of study design

This study was designed as a double-blind, placebo-controlled, mul-ticenter, randomized controlled trial. Subjectswere enrolled at two fieldcenters: Jean Mayer USDA Human Nutrition Research Center on Agingat Tufts University in Boston, MA (BOS) and Uppsala University/Karolinksa Institutet in Stockholm, Sweden (SWE). These sites werechosen, as both countries have a rapidly growing population of olderadults, which in-turn leads to an increase in the prevalence ofmobility-limitations and vitamin D insufficiency. This study was ap-proved by the Tufts Health Sciences Campus Institutional ReviewBoard, aswell as the Regional Ethical Committee of Uppsala. All enrolledparticipants participated in a moderate intensity exercise program, and

were randomized to receive a high protein, high vitamin D nutritionalsupplement or a placebo. The exercise and supplement/placebo inter-vention was 6months in duration. Outcomeswere assessed at baseline,3, and 6 months.

2.2. Eligibility

The VIVE2 eligibility criteria can be found in Table 1.

2.3. Recruitment

A total of 150 subjects were planned to be recruited across bothstudy sites (80 subjects in BOS, and 70 in SWE).

2.4. Recruitment strategies

The recruitment at BOS included a direct mailing campaign ofapproximately 400,000 letters and brochures, community-outreach

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strategies at local senior centers and senior living facilities, and localnewspaper advertisements. At SWE, the primary recruitment strategywas repeated advertisements in a local paper distributed for free inthe southern part of Stockholm County. Approximately 400 letterswere also mailed out to older adults living in the southern suburbs ofStockholm. Moreover, posters and brochures were distributed to localsenior centers and primary health care centers in the southern part ofStockholm County.

2.5. Screening

Potential participants were initially interviewed over the telephoneto assess eligibility. Those who qualify were asked to attend an orienta-tion screening visit (OSV) at the site.

The screening process was divided into two separate visits. OSV ses-sions were held 2 to 3 times per week. The OSV was conducted in agroup format, where approximately 7–15 participants would attendeach session. A brief presentation discussing the study backgroundand requirements was provided by a study coordinator. Those who de-cided to participate performed the SPPB with study staff. If participantswere eligible following the SPPB (score ≤9), a small blood samplewouldbe obtained to measure serum 25 (OH) D level (DiaSorin RIA kit(DiaSorin, Stillwater, MN, USA)). Subjects screened at SWE performedthe 400 M walk at the OSV in addition to the SPPB screening andblood sample. The 400 Mwalk was added to the OSV at SWE to addresslogistical obstacles to the local screening process, and it was agreed bythe investigators across both sites that this would not introduce any ad-ditional bias into the selection of study participants, as the same inclu-sion criteria was utilized across both sites. Subjects who are eligibleafter the OSVwere invited back to the site for evaluation of the remain-ing eligibility criteria. Subjects found to be eligible for randomizationsigned the study informed consent form prior to randomization. Asindicated in exclusion criteria in Table 1, the site PI can exclude a partic-ipant if it is believed that theymay not be appropriate for a group-basedintervention. In our previous work, participants who fall into this cate-gory are typically excluded for behavioral reasons, which may have in-fluenced the adherence of other participants, given the group-baseddesign of the study intervention. If one site was unsure about the inclu-sion of a participant, it would be discussed with the study team acrossboth sites. Though the use of this exclusion criterion is rare, the investi-gators agreed that this is important to include in a study of this design.

2.6. Informed consent

Informed consent was collected prior to each screening visit, as aseparate screening informed consent was utilized to maximize the effi-ciency of these visits. Participants were provided a copy of the informedconsent to read at home prior to each visit, and were provided ampletime to review the study details with the study staff prior to signing.In addition, a separate tissue banking informed consent was used to ob-tain consent for retaining biological samples for future unspecified use.

2.7. Study intervention

2.7.1. Physical activityAll randomized subjectswere asked to participate in an exercise pro-

gram designed by study investigators in collaboration with NestléHealth Science, Vevey, Switzerland. Participants began the interventionon a rolling basis, and class size ranged from 1 to 15 participants de-pending on the study stage (the first and last participants enrolled hada brief period of exercising alone, up to the peak where up to 15 partic-ipants would attend a class). Class sizes were not to exceed 15 partici-pants in order to ensure safety throughout each session. At least twotrained and certified interventionists supervised each class. Participantswere asked to attend three center-based exercise sessions per week,which were conducted in a supervised setting, similar to previous

interventions in this population [9]. These sessionswere used to initiatethe aerobic program and to introduce participants to the strength,stretching, and balance portions of the program in a safe and effectivemanner. The supervised setting allowed instructors to better tailor theprogram to individual needs and abilities early on, so as to preventearly dropout and to facilitate the building of self-efficacy and support,which have been found to be key to long-term physical activitymainte-nance [37,38]. These exercise sessions involved approximately 60 minof exercise instruction including 30minute aerobic, 20 minute strength(using ankle weights), a warm-up, and cool down. The strength exer-cises included chair rises, knee extension, side hip raises, knee flexion,and calf raises. The warm-up and cool down portions include flexibilityand balance exercises. Rest periods were built in between the varyingtypes of exercise throughout each session (aerobic and strength,strength and balance).

The participants were introduced to the activities of the physical ac-tivity intervention in a structured way such that they begin with lighterintensity and gradually increase intensity over thefirst 2–3weeks of theintervention. Given the limited mobility of the participants at baseline,many had to work up to completing the full 60min of exercise through-out the intervention. VIVE2 promoted walking for exercise at a moder-ate intensity and relied on ratings of perceived exertion as a method toregulate physical activity intensity. Using Borg's scale [39], that rangesfrom 6 to 20, participants were asked to walk at an intensity of 13 (ac-tivity perception SOMEWHAT HARD). They were discouraged fromexercising at levels that approach or exceed 15 (HARD) or drop to a rat-ing of 11 (FAIRLY LIGHT) or below. As participants continued to partic-ipate throughout the trial, the total amount of minutes walked at eachsessionwas gradually increasedby the study interventionists. Lower ex-tremity strengthening exercises were performed (2 sets of 10 repeti-tions) at an intensity of 15 to 16 using Borg's scale. The strengthexercises also gradually increased in intensity throughout the interven-tion period. The balance exercises were also introduced gradually. Thebattery of balance exercises were categorized into 5 levels, wherelevel 1 was the easiest, and level 5 was most difficult. Participantswere introduced to the balance exercises at level 1, andwere progressedto the more difficult levels throughout the course of the intervention.

The subjects were also encouraged to conduct physical activity out-side of class as well. The overall goal of the physical activity programwas for participants to complete at least 150 min per week of moderateintensity physical activity. This is consistent with current public healthguidelines for physical activity by older adults [40]. Subjects wereinstructed to record any physical activity performed (apart from the ex-ercise classes) on a home log provided by the study interventionists. Allparticipants were instructed not to commence any new exercise or re-habilitation program during the study, but are not instructed on levelsof voluntary or spontaneous activity.

2.7.2. Nutritional supplementThe experimental study product was a ready-to-drink, liquid

nutritional supplement in a small volume (4 fl. oz. or ca. 119 ml). Thesupplement was delivered to the sites directly from the study sponsor(Nestlé Health Science, Vevey, Switzerland) in a prepared, liquid form.At SWE, the nutritional supplement was sent monthly, directly to studyparticipant's homes. It was nutrient-dense, providing 150 kcal/serving,and provided whey protein in a proprietary concentrated form(20 g/4 fl. oz.), the daily recommended intake of vitamin D (800 IU)for older adults [41], together with a whole set of macro-andmicronutrients. The placebo was a low energy flavored drink (ca.30 kcal), not containing protein and vitamin D.

Each participant was instructed to consume one serving (4 fl. oz.)per day. The participants were either consuming the supplement orthe placebo. On exercise days, the participants would drink one bever-age immediately following the exercise class, before leaving the site.This procedure was put in place as it can impact adherence and reducethe likelihood that the participantmay forget to take it. It also provides a

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Table 2Schedule of assessments.

Assessments Screening Baseline 3-Month visit 6-Month visit

SPPB X X XMMSE XECG XHeight XWeight X X XMedications X X XVital signs X X XHistory and physicalexamination

X

400 M walk X X XPeak torque and power X XQuestionnaires X X XStair climb test X XGrip strength test X XAccelerometry X XComputed tomography X XDXA scan X XMuscle biopsy X X

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venue for participants to discuss any concerns they may have with thestudy product.

2.7.3. Adherence and retention strategiesDue to the high number of co-morbidities observed with this popu-

lation, it was very important to have a plan in place to maximize reten-tion rates. For this study, we assumed an attrition rate of approximately20%, as this is consistent with other clinical trials in this population. AtBOS, transportation was offered to study volunteers, as many oldervolunteers have difficulty commuting into the urban-based center,andmany volunteers resided in the suburban areas of Boston. Similarly,transportation programs at SWE that are publicly available were adver-tised and were utilized as needed.

In addition, this study was designed as a group-based exercise inter-vention to promote socialization, and to create a group-like atmosphere,which would assist in keeping participants, engaged and motivated toattend. In addition, several classes were offered throughout the weekin order to provide a flexible schedule. Though participants were en-couraged to adhere to a regular schedule, offering classes throughoutthe week allowed some flexibility should outside commitments arise.

In order to maximize adherence to the nutritional supplement/placebo, participants were asked to record whether or not they con-sumed their beverage each day on a study log. In addition, there was aspace to record any unintended adverse effects that may be due to thestudy product.

2.7.4. RiskAs this study was performed in a mobility-limited population,

unique measures were required to reduce risk. Though the risk ofparticipating in moderate-intensity physical activity was low, somerisks remain, such as muscle soreness, and falls. However, this studyemployed a highly rigorous screeningprocess, in order to address certainindividual's eligibility and safety for completing the exercise program. Inaddition, all exercise classes were supervised by trained and certifiedstudy staff interventionists, who thoroughly instructed participants oncorrect form, appropriate level of exertion, andwere able to assess safetyduring the study intervention. The intervention staff members wouldadjust the intensity of the exercise performed by each individual subject,to ensure that the exercises were performed effectively and safely.

Though the risks associated with the nutritional supplement werelow, it was considered very important to collect any information onany possible adverse effects. Participants were asked to record any ad-verse effects they experienced that may have been due to the studyproduct on their weekly logs, and these were reviewed regularly bythe study staff. If participants reported any adverse effects that theybelieved to be attributed to the study product, the study physicianmay decide to reduce the dose or temporarily withhold the study prod-uct to see if the side effects subside. Pending the outcome, appropriateaction would be taken by the study team.

2.8. Study measures

In order to assess the efficacy of the supplement combined withexercise, all outcomes were assessed at baseline and 6-months. More-over, in order to examine the trajectory of change among some of thefunctional outcomes, a shortened assessment was performed after3 months of the intervention. In addition, as there is a long list of out-comes for this study, the baseline visit was separated into two visits,each approximately 2–3 h in length, in order to avoid fatigue. This pro-cess was repeated at 6-months. The full schedule of assessments can befound in Table 2.

2.9. Primary study outcome

The primary study outcome was a comparison of change in 400 Mwalk performance between nutritional supplementation and placebo

(expressed as average gait speed during the 400 M walk (m/s)), after6 months of moderate intensity exercise. Change in 400 M walk timeat 3 months was considered a secondary outcome.

The 400 m walk was conducted in a long, straight hallway, wheretwo cones mark a 20 meter course. Participants were instructed towalk 10 laps around the cones at their usual pace, without over-exerting themselves. Participants must have been able to complete all10 laps in less than 15min in order to be enrolled in the trial. A straightcanemay have been used, and participantswere allowed to stop to rest,though leaning on any other object or sitting was not allowed duringthe test [42,43].

2.10. Secondary study outcomes

2.10.1. Stair climbChange in stair climb time frombaseline to 6monthswas considered

a secondary outcome. Stair climb timewas assessed using a standard setof stairs as described previously [44]. The subject was asked to ascend aset of 10 stairs as quickly as possible. Two trials were conducted with60 s of rest separating each trial. Subjects could ascend holding the rail-ing or using their assistive device if necessary. Timewas recorded usingthe Lafayette Instruments Multi-function Timer/Counter (Lafayette In-struments, Lafayette, IN). The average of the 2 attempts was recorded.

2.10.2. SPPBChange in SPPB score from baseline to 6 months was considered a

secondary outcome. The SPPB captures domains of strength, endurance,and balance and is highly predictive of subsequent disability [33]. Thetest consists of 1.) a simple test of balance, involving three differentfoot positions, that are required to be held for 10 s, 2) four meter gaitspeed, and 3) a repeated chair rise. Each component is scored using a0–4 scale and the composite score is reported from 0–12.

2.10.3. Dual Energy X-Ray Absorptiometry (DXA)Total body lean mass and composition was measured using DXA

(Hologic, Discovery A (Hologic, Bedford MA) used at BOS; GE Lunar(Madison, WI) used at SWE) [45]. Change in total body lean mass wasconsidered a secondary outcome. The DXA system generates photonsat two principal energy levels (40 and 70 KeV) which allow measure-ment of bone mineral and soft tissue. Whole body and regional fat andfat free mass were assessed for this study.

2.10.4. Computed tomography (CT)Regional changes in mid-thigh skeletal muscle cross sectional area

were assessed by CT. Scans of the non-dominant thigh were performedat the midpoint of the femur for each subject. The length of the femur

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Table 3Biological sample analysis schedule.

Time point Screening Baseline 3-Monthvisit

6-Monthvisit

BloodSerum 25 (OH) D X X X XStandard Laboratory Analytes,blood lipids, and hematology

X X X

Acute phase proteins X X XhsCRPAlbuminFibrinogen

Cytokines X X XIL-6TNF-α

Growth factors X X XIGF-1

OGTT X X XInsulinC-peptidePlasma glucose

UrineUrinalysis X

168 D.R. Kirn et al. / Contemporary Clinical Trials 43 (2015) 164–171

wasdetermined froma coronal scout image as the distance between theintercondylar notch and the trocanteric notch. All scans were obtainedusing a Siemens Somatom Scanner (Erlangen, Germany) operating at120 KV and 100 mA. Technical factors included a slice width of 10 mmand a scanning time of 1 s. All scanswere analyzed by a single investiga-tor in a blinded manner using SliceOmatic v4.2 software (Montreal,Canada). Imageswere analyzed formuscle cross sectional area, and sub-cutaneous and intermuscular fat cross sectional area. This method ofquantifying mid-thigh muscle cross sectional area has been used inmany previous trials [46–50].

2.10.5. Isokinetic peak torque and isometric strengthIsokinetic peak torque and isometric strength were determined for

the knee flexors and extensors using the Biodex System 3 IsokineticDynamometer (BiodexMedical Systems, Shirley, NY). Peak torquemea-surements were obtained with the subjects seated and the knee fullyextended. Peak torque was measured at 60, 90, 180 and 240°·sec−1.These specific angular velocities were selected because of their associa-tionwith functional performance and good reliability in this population[51]. For themeasurement of isometric strength, participants alternatedbetween extension and flexion repetitions (30 seconds rest betweeneach repetition). Participants were instructed to apply as much forceas possible for 5 s. Isometric strength for knee extensors and flexorswas considered the peak force generated across three isometric repeti-tions for each leg.

2.10.6. Hand grip strengthHand grip strength was measured in the dominant hand with a

Jamar Handheld Dynamometer (Patterson Medical, Warrenville, IL)[52]. The patients performed the test while sitting comfortably withshoulder adducted and neutrally rotated, the elbow supported on atable and flexed to 90°, and the forearm and wrist in neutral position.The patients were instructed to perform a maximal isometric contrac-tion. The test was repeated after 10 s and the highest value is recorded.

2.10.7. Mini Nutrition Assessment Short Form (MNA®-SF)TheMNA®-SF is a validated nutrition screening and assessment tool

that can identify geriatric patients age 65 and above who are malnour-ished or at risk of malnutrition [53]. Change in nutritional status basedon the MNA-SF was considered a secondary outcome.

2.10.8. Mood/depressive symptomsMood and depressive symptoms were assessed using the Center for

Epidemiologic Studies Depression Scale (CES-D). The CES-D scale is ashort self-report scale designed tomeasure depressive symptomatologyin the general population [54].

2.10.9. Quality of lifeQuality of life was assessed by employing the validatedMedical Out-

comes Study 36-item Short-Form General Health Survey (SF-36) [55].The questionnaire consists of 36 questions, is self-administered and as-sesses quality of life and wellbeing in eight multi-item scales regardingphysical functioning and perception of physical role, vitality, generaland mental health, perception of emotional role, social functioningand bodily pain.

2.11. Exploratory outcomes

2.11.1. AccelerometryTo evaluate the influence of both the nutritional supplement and the

physical activity intervention on changes in spontaneous/voluntaryphysical activity, accelerometry was performed using the ActigraphModel GT3X+ (Actigraph, Pensacola, FL) [56]. The accelerometer wasattached to an elastic belt which wraps around the subject's waist. Theaccelerometer was positioned superior to the iliac crest. Participantswere instructed to wear the accelerometer for a 7-day period during

waking hours and remove it for sleep and bathing only. Both the volumeand intensity of physical activity was quantified.

2.11.2. Insulin sensitivityA 120-minute oral glucose tolerance test (OGTT) was performed to

assess responses in insulin sensitivity to the study intervention. Mea-surements of plasma glucose, insulin and c-peptide were obtained at30 minute intervals (0, 30, 60, 90, and 120 min). Area under the curve(AUC) analysis was used to quantify changes in insulin sensitivitybetween the two treatment groups [57].

2.11.3. Muscle samplesMuscle biopsies were obtained from a subset of study participants to

investigate vitamin D molecular pathway activation when subjectsperform physical activity and consume a nutritional supplement orplacebo.

2.11.4. ComplianceCompliance with the physical activity intervention was assessed by

attendance at the scheduled physical activity sessions. A compliancelog was also given to document physical activity done in between phys-ical activity sessions. Compliance with the nutritional supplements wasmonitored using a compliance log that each subject was instructed tocomplete.

2.11.5. Biological samplesBlood samples were collected to examine standard laboratory

analytes, blood lipids and hematology, acute phase proteins, and circu-lating cytokines. A detailed analysis plan and collection schedule can befound in Table 3.

2.12. Planned statistical considerations

Theplanned sample size (N=150)will provide 90%power to detecta clinically significant difference of 0.1 m/s in average walking speedover 400 m assuming walking speed has a standard deviation of0.19m/s, Pearson correlation between baseline and follow-upmeasure-ments at least 0.5, and cumulative missingness of walking speed mea-surements no greater than 20% at 6 months.

The primary target of estimation is the mean difference in walkingspeed (m/s), derived from the 400mwalk test, attributable to nutrition-al supplementation after 6 months of moderate intensity exercise. Thiswill be estimated using a mixed-effects linear regression of walkingspeed on intervention (vs. placebo) with control for sex (a stratification

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factor) and baseline walking speed, with study center (Boston orSweden) included as a random effect. Treatment effects will be estimat-ed using treatment contrasts and associated 95% confidence intervalestimates. A secondary analysis will utilize mixed effects regressionmodels of 3 and 6 month changes in walking speed simultaneously,with participants clusteredwithin study centers. Analysis of continuoussecondary endpoints will be conducted using the same approach as de-scribed for the primary endpoint. Analysis of discrete and time-to-eventoutcomes will be developed using relevant approaches; for instance,between-group differences in incidence of fall will be assessed usingthe Kaplan–Meiermethod and proportional hazards regressionmodels.Both intention-to-treat and completer analysis will be utilized for thisstudy data. The completer analysis will include all participants whoattended at least 60% of the exercise sessions and utilized at least 60%of the planned supplementation.

2.13. Blinding

Strict measures were in place to ensure study blinding to this study.All study staff, including staff conducting the intervention, outcomes, aswell as the Principal Investigators were all blinded to treatment group(supplement vs. placebo). Randomizationwasperformed electronically,and could be broken immediately if an emergency situation arose.

2.14. Randomization

Randomization of subjects to the nutritional supplement or placebogroups was designed to stratify for gender, and utilize a separateblocked randomization schedule for males and females. Based on ourlaboratory's prior experience, it was anticipated that our sample willbe comprised of a 2:1 ratio of female to male subjects. A block size of10 was employed (allocating 5 subjects to the supplement group and5 subjects to the placebo group within each sex). A computer-generated randomization determined the order in which the supple-ments are assigned in each block. The group assignments based onthis randomization scheme were administered by a research assistantnot affiliated with this study and who had no direct contact with theresearch participants, or study staff.

3. Discussion

The primary aim of this study was to compare the effects of a highprotein, high vitamin D nutritional supplement with a placebo onexercise-induced changes in physical functioning older adults. Multiplestudies have examined the impact of protein supplementation onstrength, muscle size, and muscle quality when combined with resis-tance training in older adults [13,36,58–60]. However, this study wasthe first to examine the long term effects of a low volume ready-todrink liquid nutritional supplement providingwhey protein in a propri-etary concentrated form (20 g/4 fl. oz.) in combination with vitamin Dand other key nutrients, on 400 meter walk time in mobility-limitedolder adults.

The decision to study older individuals with demonstrated function-al limitations is multi-factorial. We have established that reduced mus-cle mass and loss of muscle strength and power are strong independentpredictors of the ability to perform several mobility-related tasks, suchas walking and stair climbing [61–65]. Targeting individuals prior tothe onset of clinically evident disability may have important publichealth implications for designing function promoting therapies toprevent mobility disability with advancing age. In addition, these “atrisk” individuals may represent a cohort of older individuals wherefunctional limitations in walking and other tasks are closely related toimpairments such as sarcopenia, reduced muscle strength and power,which may maximize the benefits from nutritional and/or exerciseinterventions.

The synergistic effect of protein supplementation on muscle func-tion following both acute and chronic exercise in young and middle-aged adults has been demonstrated [18,66]. In addition, vitamin Dsupplementation has been researched for a variety of outcomes inmany populations, including falls [67,68], fracture [69–73], and bonehealth [74]. No clinical trials have investigated the effects of protein sup-plementation combined with vitamin D supplementation on physicalactivity induced changes in older adults.

In a recent study conducted by Pahor et al. [9], it was found thatstructured, moderate intensity physical activity can prevent mobilityloss in mobility-limited older adults. This study adds further noveltyby examining the synergy of a nutritional supplement with physical ac-tivity. If the results from this study are positive, and it is shown that thenutritional supplement provides added benefit to the physical activityintervention, more specific recommendations regarding protein intakecombined with exercise may be warranted. Additionally, these resultsmay rationalize investigating the feasibility of implementing this typeof physical activity and nutritional supplementation program into acommunity setting.

The design of this study is subject to some limitations. First, as withany multi-center study, there are inherent difficulties in coordinationacross sites, particularly with sites are located in different countries.The logistics of conducting a clinical trial may differ across study sites,which can take a considerable effort to address. To overcome this obsta-cle, monthly teleconference calls were held to discuss study progress,inclusion and exclusion criteria, and other procedural difficulties thatmay have occurred at the study sites. In addition, an on-site trainingwas held at SWE, in order to ensure that procedures for all study out-comes, as well as the intervention, were conducted in a standardizedway. Further, a face-to-face meeting with the study staff from bothsites was held annually, which was used as a forum to discuss studylogistics, timelines, and he analysis plan. Second, there is limited databeing collected on nutritional status and dietary intake in this study.

In conclusion, the results of this study will further define our under-standing of the role of a nutritional supplement on whole body physicalfunctioning and accrual of fat free mass in older individuals with objec-tively measured functional limitations. Evidence from this trial willprovide further insight as to the impact of nutritional supplementationand lasting impact on physical function, body composition, and strengthamong older adults. If the nutritional supplement in this study is foundto be effective, it will in combination with physical activity provide an-other method for mobility-limited older adults to improve their healthand physical abilities.

Financial disclosures

Dr. Roger Fielding has served as a consultant to Nestle Health Sci-ence, and has received honoraria for previous speaking engagements.Professor TommyCederholmhas received speaking engagementhonor-aries from Nestle Health Science.

Acknowledgments

This work was supported by Nestlé Health Science, Vevey,Switzerland (CTA# 10.27.CLI).

This study was supported by the U.S. Department of Agriculture,under agreement No. 58-1950-0-014. Any opinions, findings, conclu-sion, or recommendations expressed in this publication are those ofthe author(s) and do not necessarily reflect the view of the U.S. Depart-ment of Agriculture.

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