Nqy 192

Effects of protein supplementation on lean body mass, muscle
strength, and physical performance in nonfrail community-dwelling

older adults: a systematic review and meta-analysis
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Dominique SM ten Haaf,1 Malou AH Nuijten,1 Martijn FH Maessen,1 Astrid MH Horstman,2 Thijs MH Eijsvogels,1 and
Maria TE Hopman1,3
1 Radboud Institute for Health Sciences, Department of Physiology, Radboud University Medical Center, Nijmegen, Netherlands; 2 FrieslandCampina,
Amersfoort, Netherlands; and 3 Division of Human Nutrition, Wageningen University, Wageningen, Netherlands
ABSTRACT Keywords: sarcopenia, elderly, protein, lean body mass, perfor-

Background: Increasing protein intake has been suggested as mance
an effective strategy to ameliorate age-related loss of muscle
mass and strength. Current reviews assessing the effect of protein
supplementation are strongly influenced by the inclusion of studies
INTRODUCTION
with frail older adults.
Objectives: We assessed the effect of protein supplementation on Age-related, progressive loss of muscle mass and strength,
lean body mass, muscle strength, and physical performance in referred to as sarcopenia, is one of the major determinants
exclusively nonfrail community-dwelling older adults. Moreover, of disability in older adults (1). The depletion of muscle
we assessed the superior effects of protein supplementation during mass predisposes older adults for bone fractures (2) and the
concomitant resistance exercise training on muscle characteristics. development of chronic metabolic diseases, such as type 2
Design: A systematic literature search was conducted on PubMed, diabetes (2, 3) and obesity (2), leading to substantial increases
Embase, and Web of Science up to 15 May 2018. We included in health care costs (4, 5).
randomized controlled trials that assessed the effect of protein To prevent or delay the age-associated decline of physical
supplementation on lean body mass, muscle thigh cross-sectional capabilities, several strategies have been proposed to counteract
area, muscle strength, gait speed, and chair-rise ability and performed the loss of muscle mass and muscle strength. Resistance exercise
random-effects meta-analyses. training is a well-established method to elicit an anabolic
Results: Data from 36 studies with 1682 participants showed no response and consequent gains in muscle mass and strength in
significant effects of protein supplementation on changes in lean older adults from the general population (6–8). Moreover, a high
body mass [standardized mean difference (SMD): 0.11; 95% CI: dietary protein intake was associated with an attenuated loss of
−0.06, 0.28], handgrip strength (SMD: 0.58; 95% CI: −0.08, 1.24), muscle mass in community-dwelling older adults (9, 10).
lower extremity muscle strength (SMD: 0.03; 95% CI: −0.20, Several systematic reviews and meta-analyses assessed the
0.27), gait speed (SMD: 0.41; 95% CI: −0.04, 0.85), or chair- effects of protein supplementation on lean body mass, muscle
rise ability (SMD: 0.10; 95%: CI −0.08, 0.28) compared with strength, and/or physical performance (11–17), but these reviews
a control condition in nonfrail community-dwelling older adults. were strongly influenced by the inclusion of studies with frail
Moreover, no superior effects of protein supplementation were
found during concomitant resistance exercise training on muscle
characteristics.
The authors reported no funding received for this study.
Conclusions: Protein supplementation in nonfrail community-
Supplemental Figures 1–7 are available from the “Supplementary data” link
dwelling older adults does not lead to increases in lean body in the online posting of the article and from the same link in the online table
mass, muscle cross-sectional area, muscle strength, or physical of contents at https://academic.oup.com/ajcn/.
performance compared with control conditions; nor does it exert Address correspondence to MTEH (e-mail: maria.hopman@
superior effects when added to resistance exercise training. Habitual radboudumc.nl).
protein intakes of most study participants were already sufficient, and Abbreviations used: DXA, dual-energy X-tray absorptiometry; RCT,
protein interventions differed in terms of type of protein, amount, randomized controlled trial; RM, repetition maximum; SMD, standardized
and timing. Future research should clarify what specific protein mean difference.
supplementation protocol is beneficial for nonfrail community- Received March 8, 2018. Accepted for publication July 16, 2018.
dwelling older adults with low habitual protein intake. Am J Clin First published online November 23, 2018; doi: https://doi.org/10.1093/
ajcn/nqy192.
Nutr 2018;108:1043–1059.
Am J Clin Nutr 2018;108:1043–1059. Printed in USA. © 2018 American Society for Nutrition. All rights reserved. 1043
1044 TEN HAAF ET AL.
older adults. For example, randomized controlled trials (RCTs) the use of the inclusion and exclusion criteria in the review
including frail older adults found beneficial effects of protein protocol (Table 1). Subsequently, the abstracts of the remaining
supplementation on muscle characteristics (18, 19), whereas studies were screened and 96 studies were assessed in full
RCTs performed in healthy older adults did not find such a text to determine whether the data could be added to the
beneficial effect (20, 21). A systematic review on the effect of meta-analysis (Figure 1). Interreviewer disagreements were
protein supplementation on muscle characteristics and physical resolved through consensus or by consulting a third reviewer
performance in community-dwelling older adults is lacking but of (MTEH). RCTs were deemed eligible if they conformed to
high relevance considering the growing population of vital older the predetermined inclusion and exclusion criteria (Table 1).
adults. “Older adults” was defined as an average age of ≥50 y.
The aim of this systematic review and meta-analysis was Moreover, participants had to be nonfrail and community-
to assess the effect of protein supplementation on lean body dwelling. Studies (or study arms) with patient populations
mass, muscle strength, and/or physical performance, in nonfrail suffering from the following diseases—cancer, muscle diseases,
community-dwelling older adults. Moreover, we aimed to lung diseases, kidney diseases, gastrointestinal diseases, diabetes,
assess the superior effect of protein supplementation during cardiovascular diseases, or immunodeficiency diseases—were

concomitant resistance exercise training. We hypothesized that excluded because of interference with muscle characteristics
protein supplementation alone does not have a beneficial effect on (24, 25). Furthermore, studies with participants that were hos-
lean body mass, muscle strength, and physical performance, but pitalized or immobilized or had an assisted-living situation were
that this may be overcome when combining resistance exercise also excluded. Suitable interventions were those studies that used
training with additional protein supplementation. oral (multinutrient) protein supplementation or a mixture of ≥8
essential amino acid or protein-rich products. The intervention
could be additional to the participants’ normal diet or replace their
METHODS normal diet. Studies in which the protein group and the control
group both performed resistance exercise training were also
Search strategy and study identification
included. Interventions with an energy intake restriction were
A systematic review was performed with the use of the Pre- excluded. The minimal duration of the intervention was set at
ferred Reporting Items for Systematic reviews and Meta-Analysis 4 wk. Studies that used ≥1 from the following outcome
statement 2015 (PRISMA) (22). The PubMed, Embase, and Web measures—lean body mass (or fat-free mass) measured with
of Science databases were systematically searched for articles dual energy X-ray absorptiometry (DXA), hydrostatic weighing
up to 15 May 2018. The following search strategy was used, (deuterium oxide dilution), whole-body air plethysmogrophy
with adaptation for each database: (Aged OR Middle aged OR (Bod Pod), or hydro densitometry (underwater weighing),
Elderly OR Old∗ people OR Old∗ person∗ OR Old∗ adult∗ OR thigh muscle cross-sectional area measured with computer-
Old∗ population∗ OR healthy older adults) AND (Protein intake ized tomography or MRI, isometric upper body and lower
OR Protein supplement∗ OR Nutritional protein∗ OR Dietary extremity muscle strength, and physical performance tests
Protein∗ OR Essential Amino Acids OR Milk Protein∗ OR that determined gait speed and chair-rise ability—were in-
Casein∗ OR Whey∗ OR Amino Acid∗) AND (Muscle strength cluded. Finally, non-English articles, conference proceedings,
AND Skeletal muscle AND Handgrip strength OR Hand strength and articles with abstracts only or study protocols only were
OR Grip strength OR leg press strength OR quadriceps strength excluded.
OR Muscle mass OR fat free mass OR lean body mass OR lean
tissue mass OR Muscle function OR Muscle quality OR physical
condition OR physical function OR physical functionality OR
physical activity OR physical active OR physical performance Data extraction
OR Physical working capacity OR physical capacity OR func- Data were extracted with the use of a predetermined data
tional performance OR Mobility condition∗ OR Mobility active extraction file. Baseline and postintervention data of lean
OR Mobility activity OR Mobility performance OR Muscles OR body mass, muscle strength, and physical performance with
Muscle OR myofibril∗ OR Muscle protein synthesis∗). To ensure corresponding SDs or SEs were independently recorded by 2
we found all the articles for our secondary aim, we performed reviewers (DSMtH and MAHN). Thigh muscle cross-sectional
an additional search in which we extended our search with area was assessed. For muscle strength, upper body and lower
the Medical Subject Headings term “AND Resistance training.” extremity strength were assessed separately. When studies
Within this search RCTs were identified with validated methods included multiple exercises to determine strength in the upper
for the different databases (23). For PubMed the sensitivity- and body or lower extremities, the exercise that measures the largest
precision-maximizing version was found most suitable, whereas muscle group was used in the analysis (e.g., upper body: lat
for Embase the Cochrane Lefebvre was used, which was also pull down > bench press > chest press > biceps curl > triceps
adapted and used for Web of Science (23). Reference lists of extension > preacher curl, respectively; and lower extremities:
included articles were manually checked for possibly eligible leg press > leg extension > leg curl, respectively). Physical
studies that were missed during the literature search (Figure 1). performance tests were divided into walking tests that determined
gait speed and therefore aerobic capacity and tests that targeted
daily functionality, e.g., chair-rise tests. When studies included
Study selection multiple walking or chair-rise tests, we used the following order
After elimination of duplicates, 2 reviewers (DSMtH and for assessing the gait speed: 400-m walk > 6-min walk > 10-
MAHN) independently screened study titles for eligibility with m walk > 6-m walk test > Short Physical Performance Battery
PROTEIN SUPPLEMENTATION IN NONFRAIL OLDER ADULTS 1045

FIGURE 1 PRISMA flowchart of search strategy outcomes. EAA, essential amino acids; PRISMA, Preferred Reporting Items for Systematic Reviews
and Meta-Analysis.
gait speed > 4 square step test, respectively; and for chair- Assessment of risk of bias in included studies
rise tests: TUG (Timed Up-and-Go) test > Short Physical
Performance Battery chair-rise test 30-s sit-to-stand test > 5 The quality of each eligible study was independently assessed
times sit-to-stand test > 15-s step test, respectively. Other by 2 reviewers (DSMtH and MAHN), with the use of the
information gathered included publication year, sample size, Downs and Black checklist (26) which is composed of 27
participants’ sex, age, BMI, and intervention details: duration, items for evaluating the risk of bias, based on the quality
type of protein, amount, type of placebo, training frequency, of reporting, external validity, internal validity (bias), internal
type of training, and training intensity. When viable information validity (confounding/selection bias), and power. In total 32
(i.e., information about the results needed for inclusion in our points can be received with the original Downs and Black
meta-analysis) was missing, an attempt was made to request checklist of 27 questions. We excluded the final question
missing information from the authors by email (n = 14 studies; (question 27) about power, because we performed a meta-analysis
authors of n = 6 studies provided requested information). For in which the independent power of the studies is irrelevant and
5 included studies results were depicted in figures, thus the underpowered studies will already be given less weight in the
data were extracted with the use of GetData Graph Digitizer pooled analysis. On this excluded question 0–5 points could be
software version 2.26. Two studies could still be included received, therefore, our modified index could result in a score
in some analyses, but not all of their outcomes could be of 0–27. The quality scores were calculated and ranked on
included in the analyses because some viable information was a 4-category scale: poor (<15), moderate (15–19), good (20–
missing. 24), and excellent (≥25). Studies that were assessed as “poor”
TABLE 1
Inclusion and exclusion criteria
Inclusion criteria Exclusion criteria

Population
- Mean age of >50 y - Hospitalized patients/immobilized individuals (bed rest, cast)
- Community dwelling - Assisted-living situation
- Nonfrail older adults - Participants with cancer, muscle diseases, lung diseases, kidney
diseases, gastrointestinal diseases, diabetes, cardiovascular diseases,
or immunodeficiency diseases
Intervention
- All randomized controlled trials that used oral (multinutrient) protein or - Amino acid mix with <8 essential amino acids
essential amino acid supplementation/nutritional products of any consistency
- Oral protein or amino acid supplementation can be additional to the - Dietary counseling only
participants’ normal diet or replace their normal diet
- Effect of protein or amino acid supplementation is assessed and compared with - Intervention with energy intake restriction

a (different) control group
- Studies in which the protein or amino acid group and the control group - Nonoral feeding
both performed resistance exercise training are also included.
The supplementation should be consumed ≥3 times/wk (around resistance
exercise training).
- Minimal duration of 4 wk
Outcome
- Lean body mass or fat-free mass
- Thigh muscle cross-sectional area
- Upper body or lower extremity muscle strength in kilograms
- Physical performance (gait speed or chair-rise test)
Other
- English language - Abstract only
- Full papers - Conference proceedings
- Study protocol/Letter to the editor
quality were excluded from the analysis. The Downs and Black control group. Analyses to assess the following comparisons—
checklist has been tested and found positive in terms of internal 1) protein supplementation with control condition and 2) protein
consistency, face validity, content validity, criterion validity, and supplementation during concomitant resistance exercise with
reproducibility. Only construct validity was not accomplished, control condition with resistance exercise—were performed.
something not achieved by most tools (27). The Downs and Black Forest plots were generated to illustrate the study-specific effect
checklist has demonstrated good inter-rater reliability (r = 0.75) sizes along with a 95% CI and for each separate analysis
and test-retest reliability (r = 0.88) (26). Cohen’s kappa (κ) the average duration of the intervention per participant was
values for the inter-rater agreement between the 2 reviewers (28) calculated in weeks. Cochran’s Q statistic and the I2 statistic were
were determined to be 0.85. This means a “strong” strength of calculated to assess the degree of heterogeneity across studies.
agreement (28). Interreviewer disagreements were resolved by The Q statistic indicates statistically significant heterogeneity at
consensus. P < 0.10. The I2 statistic reflects the percentage of the observed
between-study variability. An I2 >50% is considered to represent
substantial heterogeneity (32). Publication bias was assessed via
visual analysis of the funnel plot asymmetry with the use of the
Data synthesis and analysis “trim and fill” and “Classic fail ‘n safe” algorithms. To assess
whether any individual studies included in the meta-analysis had
To account for potential heterogeneity between studies, a
a disproportionate effect on the results, sensitivity analysis was
random-effects model (specified a priori) was used to determine
performed via the “remove-one” analysis. All calculations and
the overall effect size of the intervention (protein supple-
plots were performed in CMA-2 (Comprehensive Meta-analysis
mentation) on lean body mass, muscle strength, and physical
version 2, 2011, Biostat, Englewood, NJ).
performance. For each outcome measure of interest, a meta-
analysis was performed to determine the pooled effect size in
terms of the standardized mean difference (SMD) with corre-
sponding 95% CI. A correlation of 0.5 between the outcomes RESULTS
measured in each study arm (i.e., protein compared with control)
was assumed (29, 30). The magnitude of the SMD can be Selection of studies for the meta-analysis
interpreted as small, SMD = 0.2; medium, SMD = 0.5; and large, The original search resulted in 2708 studies. Twelve additional
SMD = 0.8 (31). When a study contained 2 protein intervention studies were found from the reference lists of the included full-
study arms (n = 5 studies), both study arms were included text papers. After removal of duplicates and elimination of papers
in the study analysis and individually compared against the based on the eligibility criteria, 36 studies were included of which
31 studies presented unique data not shown in other studies (mean ± SD: 5.8 ± 1.1, 83% of total); however, most of the
included. A total of 11 studies assessed lean body mass, upper studies scored low on external validity (1.7 ± 0.8, 57% of total).
body strength, lower body strength, gait speed, and/or chair-rise The quality of the majority of the studies was “moderate” or
ability. Moreover, 18 studies assessed the additional effect of “good” (50% and 42%, respectively) and 3 studies were rated
protein intake compared with controls on these variables while “excellent” (8%). The mean ± SD total score on the Downs and
performing resistance exercise training. One study compared the Black checklist was 19.6 ± 3.2 out of 27 (Table 2).
effect of protein supplementation in 4 groups: protein compared
with controls in groups without concomitant resistance exercise
training and protein compared with controls in groups during Cohort characteristics
concomitant resistance exercise training (33); and another study
assessed the effect of protein supplementation first without re- Data from 1682 participants were included in the analyses:
sistance training and subsequently during concomitant resistance 768 participants in studies that assessed protein supplementation
training (34) (Figure 1). Included studies were published from compared with a control condition, 865 participants in studies
1992 to 2018. that assessed the additional effect of protein intake compared
with a control condition during concomitant resistance

exercise training, and in 49 participants the effect of protein
supplementation was assessed and thereafter the effect of protein
Quality assessment supplementation during concomitant resistance exercise training
Thirty-six studies were included in the quality assessment. compared with a control condition (Table 3) (20, 21, 33–66).
The studies scored moderate to good on internal validity (bias) Eleven studies exclusively included male subjects, whereas in
TABLE 2
Quality of included studies, based on the Downs and Black checklist (26)
Reporting External validity Confounding Total score

(maximum (maximum Bias (maximum (maximum (maximum Quality as per cutoff
Authors (ref) score = 11) score = 3) score = 7) score = 6) score = 27) described
Aleman-Mateo et al. (35) 10 3 5 6 24 Good
Arnarson et al. (36) 8 3 7 6 24 Good
Bell et al. (34) 8 2 6 5 21 Good
Bemben et al. (37) 5 1 7 5 18 Moderate
Campbell et al. (38) 6 1 7 4 18 Moderate
Campbell et al. (39) 5 1 7 4 17 Moderate
Candow et al. (20) 5 1 7 3 16 Moderate
Carter et al. (40) 6 1 7 6 20 Good
Castaneda et al. (41) 8 0 5 4 17 Moderate
Chanet et al. (42) 11 2 7 6 26 Excellent
Daly et al. (43) 9 2 3 4 18 Moderate
Dillon et al. (44) 7 1 6 4 18 Moderate
Eliot et al. (45) 6 1 6 4 17 Moderate
Farnfield et al. (46) 6 1 6 3 16 Moderate
Godard et al. (47) 7 1 4 3 15 Moderate
Holm et al. (48) 6 3 7 4 20 Good
Iglay et al. (50) 7 2 5 4 18 Moderate
Iglay et al. (49) 8 2 4 3 17 Moderate
Ispoglou et al. (51) 4 2 7 2 15 Moderate
Kawada et al. (52) 6 1 6 2 15 Moderate
Kerstetter et al. (53) 9 2 7 5 23 Good
Kukuljan et al. (33) 10 2 4 4 20 Good
Leenders et al. (54) 5 1 6 5 17 Moderate
Markofski et al. (55) 8 1 7 5 21 Good
Meredith et al. (56) 7 1 5 3 16 Moderate
Mitchell et al. (58) 7 1 5 3 16 Moderate
Mitchell et al. (57) 10 3 5 6 24 Excellent
Nabuco et al. (59) 8 3 6 6 23 Good
Norton et al. (60) 9 2 5 5 21 Good
Rossato et al. (61) 10 1 5 3 19 Moderate
Scognamiglio et al. (62) 9 2 7 5 23 Good
Seino et al. (63) 11 3 6 6 26 Excellent
Thomson et al. (64) 8 3 6 5 22 Good
Torres et al. (65) 10 2 4 4 20 Good
Verdijk et al. (21) 8 1 7 5 21 Good
Zhu et al. (66) 9 3 5 6 23 Good
TABLE 3
Overview of the characteristics of the included studies1
1048
Habitual protein Protein and placebo supplementation

intake,
Placebo Iso
g · kg−1 · d−1
Authors (ref) Group n (M/F) Age, y BMI, kg/m2 Duration, wk Type of protein Amount, g/d type caloric Timing
Aleman-Mateo et al. (35) Prot 49 (25/24) 70.8 ± 7.6 26.9 ± 3.3 12 NR Ricotta 18 — — B, L, D
Con 49 (24/25) 69.6 ± 6.4 27.3 ± 3.8 12 NR — — NP NP —
Arnarson et al. (36)2 Prot 83 73.3 ± 6.0 28.1 ± 4.4 12 1.00 ± 0.26 Whey 20 (3 d/wk) — — AE
Con 78 74.6 ± 5.8 29.4 ± 4.8 12 0.92 ± 0.30 — — CHO Y AE
Bell et al. (34)2 Prot 25 (25/0) 71 (1) 28.9 (0.8) 6 excl. 1.1 (0.3) Whey (+ 30 — — B, BBT
training + 12 creatine + calcium + vit
incl. training D + n–3 PUFA)
Con 24 (24/0) 74 (1) 28.1 (0.7) 6 excl. 1.2 (0.3) — — CHO NR B, BBT
training + 12
incl. training
Bemben et al. + Carter et al. + Prot 11 (11/0) 58.2 (2.0) 28.63 14 0.95 ± 0.5 Whey 35 (3 d/wk) — — AE
Eliot et al. (37, 40, 45)2
Con 10 (10/0) 56.1 (1.4) 31.33 14 0.94 ± 0.5 — — CHO N AE
Campbell et al. + Campbell Prot 6 (5/1) 64 (2) 26.53 14 NR Lactovovegetarian diet incl. 1253 ,4 — — NA
et al. (38, 39)2 milk-based beverages
Con 6 (3/3) 66 (4) 25.53 14 NR Lactovovegetarian diet incl. 623 , 4 NA N NA
milk-based beverages
Candow et al. (20)2 Prot1 9 (9/0) 63.3 (1.1) 28.23 12 NR Protein 26.33 (3 d/wk) — — BE
Prot2 10 (10/0) 66.5 (1.7) 28.53 12 NR Protein 25.63 (3 d/wk) — — AE
Con 10 (10/0) 64.6 (1.3) 29.13 12 NR — — CHO NR BE, AE
Castaneda et al. (41) Prot 6 (0/6) 72 (1) 26.4 ± 1.7 9 NR Diet incl. milk-based 60.64 — — NA
beverages
Con 6 (0/6) 71 (2) 27.3 ± 1.1 9 NR Diet incl. milk-based 31.74 NA N NA
beverages
Chanet et al. (42) Prot 12 (12/0) 70.3 ± 4.3 24.4 ± 3.3 6 1.3 ± 0.3 Whey (+ leucine + 20 — — BB
vit D)
TEN HAAF ET AL.
Con 12 (12/0) 70.8 ± 3.5 25.1 ± 2.5 6 1.3 ± 0.2 — — NR N BB

Daly et al. + Torres et al. (43, Prot 53 (0/53) 72.1 ± 6.4 27.7 ± 3.9 16 1.07 ± 0.375 Lean red meat 45 (6 d/wk) — — 6 d/wk
65)2
Con 47 (0/47) 73.6 ± 7.7 27.6 ± 4.8 16 1.13 ± 0.40 — — CHO N 6 d/wk
Dillon et al. (44) Prot 7 (0/7) 67 ± 1 26.93 12 NR EAA 15 — — MAS
Con 7 (0/7) 69 ± 3 26.83 12 NR — — Lactose NR MAS
Farnfield et al. (46)2 Prot 9 (9/0) 68.1 ± 1.6 27.5 ± 0.8 12 1.5 ± 0.1 Whey 26.6 (3 d/wk) — — AER
Con 9 (9/0) 67.4 ± 1.3 27.5 ± 1.0 12 1.1 ± 0.1 — — NR N AER
Godard et al. (47)2 Prot 8 (8/0) 70.8 (1.5) 29.03 12 1.13 EAA + CHO 12 — — AER
Con 9 (9/0) 72.1 (1.9) 24.33 12 1.13 — — NP N —
Holm et al. (48)2 Prot 13 (0/13) 55 (1) 24 (1) 24 1.043 Whey (+ 10 (3 d/wk) — — AE
CHO + calcium + vit D)
Con 16 (0/16) 55 (1) 27 (1) 24 0.893 — — CHO N AE
Iglay et al. + Iglay et al. (49, 50)2 Prot 18 (8/10) 61 (2) 26.7 (0.9) 12 1.15 ± 0.175 Omnivorous diet 884 — — NA
Con 18 (9/9) 62 (2) 25.6 (0.8) 12 1.11 ± 0.345 Omnivorous diet 644 NA Y NA
Ispoglou et al. (51) Prot1 8 (3/5) 71.1 ± 2.7 26.6 ± 3.8 12 0.95 ± 0.315 EAA 15 (20% LEU) — — MEV
Prot2 8 (4/4) 71.9 ± 3.0 26.7 ± 3.4 12 1.06 ± 0.315 EAA 15 (40% LEU) — — MEV
Con 9 (4/5) 71.8 ± 2.7 26.3 ± 3.9 12 1.10 ± 0.345 — — CHO Y MEV
Kawada et al. (52)2 Prot1 10 (5/5) 65 ± 1 22.63 26 1.473 EAA 3 — — B
Prot2 11 (3/8) 67 ± 3 22.83 26 1.333 EAA 6 — — B, D
Con 8 (4/4) 70 ± 1 22.93 26 1.333 — — CHO NR B
Kerstetter et al. (53) Prot 106 (17/89) 69.9 ± 6.1 26.1 ± 3.4 78 1.07 ± 0.03 Whey protein isolate 45 — — NR
Con 102 (13/89) 70.5 ± 6.4 26.4 ± 4.0 78 1.06 ± 0.03 — — CHO Y NR
(Continued)

TABLE 3
(Continued)
Habitual protein Protein and placebo supplementation
intake,
Placebo Iso
g · kg−1 · d−1
Authors (ref) Group n (M/F) Age, y BMI, kg/m2 Duration, wk Type of protein Amount, g/d type caloric Timing
Kukuljan et al. (33) Prot1 43 (43/0) 61.7 ± 7.6 27.4 ± 3.7 78 1.26 ± 0.32 Milk protein (with calcium 13.2 — — B, R
and vit D)
Prot2 43 (43/0) 61.7 ± 7.7 27.7 ± 3.3 78 1.23 ± 0.28 Milk protein (with calcium 13.2 — — B, R
and vit D)
Con1 44 (42/0) 60.7 ± 7.1 28.1 ± 3.3 78 1.32 ± 0.32 — — NP NP —
Con2 42 (42/0) 59.9 ± 7.4 26.7 ± 2.9 78 1.33 ± 0.31 — — NP NP —
2
Leenders et al. (54) Prot 27 (15/12) M: 70 (1) F: M: 27.2 (0.7); 24 M: 1.1 (0.1) F: Milk protein 15 — — B
72 (2) F: 24.2 (0.7) 1.2 (0.1)
Con 26 (14/12) M: 70 (1) F: M: 26.7 (0.6); 24 NR — — CHO N B
69 (1) F: 25.0 (0.4)
Markofski et al. (55) Prot 13 (NR) Range: 26.5 (1.0) 24 0.95 ± 0.04 EAA 15 — — BM
65–85
Con 11 (NR) Range: 28.0 (0.8) 24 NR — — CHO NR BM
65–85
2 3
Meredith et al. (56) Prot 6 (6/0) 67.8 ± 1.1 24.8 ± 0.8 12 1.24 Protein (+ 20 — — MAS
CHO + Fat + vit + min)
Con 5 (5/0) 64.8 ± 2.3 25.4 ± 0.9 12 1.213 — — NP NP —
Mitchell et al. (58)2 Prot 8 (8/0) 74.4 ± 5.4 26.9 ± 3.2 12 NR Protein 14 — — B or AE
Con 8 (8/0) NR NR 12 NR — — CHO Y B or AE
Mitchell et al. (57) Prot 14 (14/0) 73.7 ± 3.3 28.2 ± 3.3 10 1.1 ± 0.3 Omnivorous diet 1364 — — NA
Con 15 (15/0) 74.7 ± 3.9 28.4 ± 5.1 10 1.2 ± 0.4 Omnivorous diet 744 NR Y NA
Nabuco et al. (59)2 Prot1 24 (0/24) 67.5 ± 5.2 26.4 ± 5.2 12 0.92 ± 0.20 Whey 27.1 (3 d/wk) — — BE
Prot2 23 (0/23) 66.2 ± 9.4 25.3 ± 5.4 12 0.94 ± 0.36 Whey 27.1 (3 d/wk) — — AE
Con 23 (0/23) 66.5 ± 7.2 23.8 ± 3.7 12 0.95 ± 0.27 — — CHO Y BE, AE
Norton et al. (60) Prot 31 (9/22) 62.2 ± 4.7 25.7 ± 3.1 24 1.2 ± 0.3 Milk protein 123 — — B, L
Con 29 (5/24) 59.5 ± 5.8 25.9 ± 4.1 24 1.2 ± 0.3 — — CHO Y B, L
Rossato et al. (61)2 Prot 11 (0/11) 63.4 ± 7.6 28.1 ± 5.5 10 0.82 ± 0.29 Omnivorous diet 774 — — NA
Con 12 (0/12) 63.0 ± 8.6 28.4 ± 6.0 10 0.78 ± 0.30 Omnivorous diet 594 NA Y NA
Scognamiglio et al. (62) Prot 48 (20/28) 74 ± 6 27.4 ± 3.6 12 NR AA (+ CHO) 36 — — MANS
Con 47 (22/25) 74 ± 5 27.3 ± 3.7 12 NR — — CHO N MANS
Seino et al. (63)2 Prot 40 (6/34) 73.4 ± 4.3 22.9 ± 2.9 12 1.39 ± 0.36 Milk protein (+ 10.5 — — L
micronutrients)
Con 40 (7/33) 73.7 ± 4.3 22.9 ± 2.2 12 1.28 ± 0.26 — — NA NA NA
Thomson et al. (64)2 Prot1 54 (25/29) 61.3 ± 6.9 27.7 ± 3.9 12 1.06 ± 0.10 Dairy 27 — — AE
PROTEIN SUPPLEMENTATION IN NONFRAIL OLDER ADULTS
Prot2 64 (29/35) 61.7 ± 8.3 27.5 ± 3.7 12 1.08 ± 0.09 Soy 27 — — AE

Con 61 (27/34) 61.5 ± 6.9 27.6 ± 3.3 12 1.02 ± 0.05 — — CHO Y AE
Verdijk et al. (21)2 Prot 13 (13/0) 72 ± 2 26.5 ± 1.0 12 1.1 ± 0.1 Casein hydrolysate 20 (3 d/wk) — — BE, AE
Con 13 (13/0) 72 ± 2 27.4 ± 1.1 12 1.1 ± 0.1 — — NR NR BE, AE
Zhu et al. (66) Prot 101 (0/101) 74.2 ± 2.8 26.1 ± 3.8 104 1.2 ± 0.3 Milk protein 30 — — BB
Con 95 (0/95) 74.3 ± 2.6 27.2 ± 4.0 104 1.1 ± 0.3 — — CHO Y BB
1
n = 36. Values are means ± SDs or means (SEs).Resistance exercise training without protein supplementation not incorporated. AA, amino acids; AE, after exercise; AER, after exercise or at random; B, breakfast; BB, before
breakfast; BBT, before bed time; BE, before exercise; BM, between meals; CHO, carbohydrate; Con, control; D, dinner; EAA, essential amino acids; excl., excluding; incl., including; L, lunch; LEU, leucine; MANS, morning, afternoon, and
night snack; MAS, morning and afternoon snack; MEV; supplement alongside food, morning and evening; N, no; NA, not applicable; NP, no placebo; NR, not reported; Prot, protein; R, random; ref, reference; vit D, vitamin D; Y, yes.
2
Studies with a concomitant resistance exercise intervention.
3
Calculated from average weight (and height).
4
Total daily protein intake.
5
Requested data from authors.
1049

7 studies, female subjects were exclusively included. Thirteen condition (SMD = 0.11; 95% CI: −0.06, 0.28, P = 0.19) with
studies included both sexes. The average habitual protein intake no heterogeneity (Figure 2) after 41 ± 32 wk.
was below the protein recommendation of 0.8 g · kg−1 · d−1
in 1 study (61) (Table 3). Protein intervention strategies were
heterogeneous and included 1) protein supplements [i.e., whey Muscle strength
or (milk) protein, n = 11; or essential amino acids, n = 6], 2) Seven out of 9 studies assessing the effect of protein
multi-ingredient-nutrient supplements with protein and other supplementation on upper body strength focused on handgrip
nutrients such as micronutrients or omega fatty acids (n = 6), strength. Handgrip strength tended to increase more after 47 ± 45
3) food products with high protein content (i.e., ricotta, lean red wk of protein supplementation compared with the control
meat, dairy, or soy) (n = 3), or 4) a diet with a high protein intake condition (SMD: 0.58; 95% CI: −0.08, 1.24, P = 0.08) with
compared with a diet with a low protein intake (n = 5) (Table 3). significant heterogeneity (Figure 3A). No differences in the
With specific reference to the type of resistance exercise training changes in lower extremity strength were observed between
performed, 17 studies performed whole-body resistance exercise protein supplementation and control conditions after 90 ± 20 wk
training for 2–3 d/wk and 3 studies performed leg exercise for 3 (SMD: 0.03; 95% CI −0.20, 0.27, P = 0.78) (Figure 3B), and

d/wk. The intensity of the exercise training ranged between 50% heterogeneity was absent.
and 85% of 1 repetition maximum (RM) (Table 4).
Physical performance
Effect of protein supplementation Gait speed tended to increase more after 31 ± 30 wk of protein
supplementation compared with control conditions (SMD: 0.41;
Lean body mass 95% CI: −0.04, 0.85, P = 0.08), with significant heterogeneity
Ten studies assessed the effect of protein supplementation (Figure 4A). No difference in chair-rise time was observed after
compared with a control condition on lean body mass with the 58 ± 42 wk of protein supplementation compared with control
use of DXA. Protein supplementation was not associated with conditions (SMD: 0.10; 95% CI: −0.08, 0.28, P = 0.26), with no
greater changes in lean body mass compared with the control heterogeneity present (Figure 4B).
TABLE 4
Overview of the resistance exercise training characteristics of the included studies with a resistance exercise intervention1
Training
Type of
Authors (ref) Habitual exercise d/wk training RT intensity Training volume
Arnarson et al. (36) 82% regular PA, 67% ≥30 min/d 3 WBR 75–80% 1RM 3 sets of 6–8 reps
Bell et al. (34) NR 3 WBR 65–80% 1RM 3 sets of 10–12/6–8 reps
Bemben et al. + Carter et al. + Eliot et al. NR 3 WBR 80% 1RM 3 sets of 8 reps
(37, 40, 45)
Campbell et al. + Campbell et al. (38, 39) NR 3 WBR 80% 1RM 2–3 sets of 8–12 reps
Candow et al. (20) NR 3 WBR 70% 1RM 3 sets of 10 reps
Daly et al. + Torres et al. (43, 65) Prot: 9.3 ± 5.6 h/wk 2 WBR 14–16 Borg scale 3 sets of 8–12 reps
Con: 8.1 ± 4.0 h/wk
Farnfield et al. (46) NR 3 WBR 50–80% 1RM 2 sets
Godard et al. (47) NR 3 Knee 80% 1RM 3 sets of 10–volitional exhaustion reps
extension
Holm et al. (48) Prot: 2 persons ran 1x/wk 2–3 WBR 10–20 rep max 3–5 sets of 8–15 reps
Con: 3 persons ran 1x/wk
Iglay et al. + Iglay et al. (49, 50) Prot: 55 ± 7 units/wk 3 WBR 80% 1RM 3 sets of 8–volitional exhaustion reps
Con: 57 ± 7 units/wk
Kawada et al. (52) >23 exercises/wk (3 MET-20 min) 2 WBR 30% 1RM 2 sets of 20–30 reps + 10 min standing
cycling at 100 Watt
Kukuljan et al. (33) Prot1: 3.7 ± 3.9 hr/wk 3 WBR 50–85% 1RM 2–3 sets of 8–12 reps
Prot2: 3.3 ± 3.8 hr/wk
Con1: 3.6 ± 3.4 hr/wk
Con2: 3.4 ± 4.1 hr/wk
Leenders et al. (54) M: 1.48 ± 0.19 MET-h/d 3 WBR 60–80% 1RM 3–4 sets of 8–15 reps
F: 1.44 ± 0.15 MET-h/d
Meredith et al. (56) NR 3 Upper leg 80% 1RM 3 sets of 8 reps
Mitchell et al. (58) NR 3 WBR 75% 1RM 3–4 sets
Nabuco et al. (59) NR 3 WBR 60–70% 1RM 3 sets of 8–12 reps
Rossato et al. (61) NR 3 WBR 70% 1RM 1–6 sets of 8–12 reps
Seino et al. (63) Prot: 61.7 ± 31.3 MET-h/wk 2 WBR NA 2 sets of 20 reps
Con: 66.4 ± 44.4 MET-h/wk
Thomson et al. (64) NR 3 WBR 8 rep max 1–3 sets of 8–20 reps
Verdijk et al. (21) NR 3 Leg 60–80% 1RM 4 sets of 8–15 reps
1
n = 16. Values are means ± SDs. Con, control; max, maximum; MET, Metabolic Equivalent of Task; NA, not applicable; NR, not reported; PA, physical activity; Prot,
protein; RM, repetition maximum; rep, repetition; RT, resistance exercise training; WBR, whole-body resistance exercise training.
Study Relative weight SMD (95% CI) P value
Aleman-Mateo, 2014 (35) 17.9 0.06 (-0.34, 0.45) 0.77

Bell, 2017 (34) 8.7 0.44 (-0.13, 1.00) 0.13
Castaneda, 1995 (41) 2.1 0.68 (-0.48, 1.85) 0.25
Chanet, 2017 (42) 4.4 0.10 (-0.70, 0.90) 0.80
Dillon, 2009 (44) 2.5 0.19 (-0.86, 1.24) 0.72

Ispoglou, 2016 Supplement with 20% leucine (51) 3.1 -0.05 (-1.00, 0.90) 0.92
Ispoglou, 2016 Supplement with 40% leucine (51) 3.1 0.01 (-0.94, 0.96) 0.98
Kerstetter, 2015 (53) 22.1 0.08 (-0.28, 0.44) 0.66
Kukuljan, 2009 (33) 16.2 0.05 (-0.37, 0.48) 0.80
Markofski, 2018 (55) 4.4 0.05 (-0.37, 0.48) 0.80
Mitchell, 2017 (57) 5.2 0.27 (-0.47, 1.00) 0.48
Norton, 2016 (60) 11.0 0.01 (-0.50, 0.51) 0.98

Overall 0.11 (-0.06, 0.28) 0.19
-3.00 -1.50 0.00 1.50 3.00
Favors control Favors protein
FIGURE 2 Forest plots of the SMD of protein supplementation compared with a control condition on total lean body mass. For each trial, the black square
represents the point estimate of the intervention effect. The horizontal line joins the lower and upper limits of the 95% CI of this effect. The area of the black
square represents the relative weight of the study in the meta-analysis. The black diamond represents the pooled estimate result (SMD) of the random-effects
meta-analysis. No heterogeneity was present (Cochran’s Q = 2.92; I2 = 0.0%, P = 0.99). SMD, standardized mean difference.
Effect of protein supplementation during resistance exercise assessed in 15 studies of which 11 studies used DXA (21, 33, 34,
training 36, 37, 40, 43, 45, 48, 54, 61, 63, 64), 2 studies hydrostatic weigh-
Lean body mass ing (deuterium oxide dilution) (38, 39, 49, 50), 1 study measured
with the Bod Pod (20), and another study determined body
The additional effect of protein supplementation during composition with hydro densitometry (underwater weighing)
concomitant resistance exercise training on lean body mass was (56). Three of these included study arms reported fat-free mass
A Study Relative weight SMD (95% CI) P value
Aleman-Mateo, 2014 (35) - Handgrip 14.2 0.08 (-0.32, 0.47) 0.70

Castaneda, 1995 Right arm (41) - Handgrip 10.1 0.87 (-0.31, 2.06) 0.15
Chanet, 2017 (45) - Handgrip 12.2 0.39 (-0.42, 1.20) 0.35
Ispoglou, 2016 Supplement with 20% leucine (51) - Handgrip 11.4 0.28 (-0.68, 1.23) 0.57
Ispoglou, 2016 Supplement with 40% leucine (51) - Handgrip 11.4 0.16 (-0.80, 1.11) 0.75
Mitchell, 2017 (58) - Handgrip 12.6 0.18 (-0.55, 0.91) 0.62
Scognamiglio, 2005 (62) - Handgrip 13.6 2.52 (1.99, 3.06) <0.01
Zhu, 2015 (66) - Handgrip 14.5 0.11 (-0.18, 0.40) 0.45
Overall 0.58 (-0.08, 1.24) 0.08
-3.00 -1.50 0.00 1.50 3.00
B Study Relative weight SMD (95% CI) P value
Castaneda, 1995 (41) - Leg extension 4.3 0.35 (-0.79, 1.49) 0.54
Kukuljan, 2009 (33) - Leg press 30.6 0.00 (-0.42, 0.43) 1.00
Zhu, 2015 (66) - Leg extension 65.1 0.03 (-0.26, 0.32) 0.86
Overall 0.03 (-0.20, 0.27) 0.78
-3.00 -1.50 0.00 1.50 3.00
FIGURE 3 Forest plots of the SMD of protein supplementation compared with a control condition on upper body (A) and lower extremity (B) muscle
strength. For each trial, the black square represents the point estimate of the intervention effect. The horizontal line joins the lower and upper limits of the 95%
CI of this effect. The area of the black square represents the relative weight of the study in the meta-analysis. The black diamond represents the pooled estimate
result (SMD) of the random-effects meta-analysis. Whereas significant heterogeneity was present (Cochran’s Q = 67.0; I2 = 89.6%, P < 0.001) in the analysis
of the upper body (A), no heterogeneity was present (Cochran’s Q = 0.33; I2 = 0.0%, P = 0.85) in the analysis of the lower extremity (B). SMD, standardized
mean difference.
A Study Relative weight SMD (95% CI) P value
Aleman-Mateo, 2014 (35) - SPPB gait speed 15.4 0.19 (-0.20, 0.59) 0.34
Bell, 2017 (34) - 6 min walk 12.9 -0.28 (-0.92, 0.36) 0.39
Chanet, 2017 (42) - SPPB gait speed 11.1 0.50 (-0.31, 1.31) 0.23
Ispoglou, 2016 Supplement with 20% leucine (51) - 6 min walk 9.7 0.34 (-0.62, 1.30) 0.48
Ispoglou, 2016 Supplement with 40% leucine (51) - 6 min walk 9.7 0.26 (-0.69, 1.22) 0.59
Kukuljan, 2009 (33) - 6 m walk test 15.1 0.32 (-0.11, 0.74) 0.15
Markofski, 2018 (55) - 400 m walk test 11.2 0.18 (-0.63, 0.98) 0.67
Scognamiglio, 2005 (62) - 6 min walk 14.8 1.55 (1.09, 2.01) <0.01
Overall 0.41 (-0.04, 0.85) 0.08
-3.00 -1.50 0.00 1.50 3.00

B Study Relative weight SMD (95% CI) P value
Aleman-Mateo, 2014 (35) - SPPB chair-rise test 20.1 0.07 (-0.33, 0.46) 0.74

Bell, 2017 (34) - TUG 7.7 0.32 (-0.32, 0.96) 0.32
Chanet, 2017 (42) - SPPB chair-rise test 4.8 0.38 (-0.43, 1.19) 0.36
Ispoglou, 2016 Supplement with 20% leucine(51) - 30s sit-to-stand 3.5 0.16 (-0.79, 1.11) 0.74
Ispoglou, 2016 Supplement with 40% leucine(51) - 30s sit-to-stand 3.4 0.44 (-0.52, 1.40) 0.37
Kukuljan, 2009 (33) - 15s step test 17.4 -0.07 (-0.49, 0.36) 0.76
Mitchell, 2017 (57) - TUG 5.9 0.12 (-0.61, 0.85) 0.75
Zhu, 2015 (66) - TUG 37.1 0.08 (-0.21, 0.37) 0.60
Overall 0.10 (-0.08, 0.28) 0.26
-3.00 -1.50 0.00 1.50 3.00
FIGURE 4 Forest plots of the SMD of protein supplementation compared with a control condition on gait speed (A) and chair-rise performance (B). For
each trial, the black square represents the point estimate of the intervention effect. The horizontal line joins the lower and upper limits of the 95% CI of this
effect. The area of the black square represents the relative weight of the study in the meta-analysis. The black diamond represents the pooled estimate result
(SMD) of the random-effects meta-analysis. Whereas significant heterogeneity was present (Cochran’s Q = 29.65; I2 = 76.4%, P < 0.001) in the analysis of
gait speed (A), no heterogeneity was present (Cochran’s Q = 2.06; I2 = 0.0%, P = 0.96) in the analysis of chair-rise performance (B). SMD, standardized
mean difference; SPPB, Short Physical Performance Battery; TUG, Timed Up-and-Go test.
rather than lean body mass (37, 39, 56). Protein supplementation Physical performance
during resistance exercise training resulted in no significant larger Gait speed tended to increase more with protein
effect on lean body mass compared with controls receiving solely supplementation compared with the control condition, whereas
resistance exercise training after 23 ± 25 wk (SMD: 0.08; 95% no differences were observed in chair-rise time, after 26 ± 26 wk
CI: −0.06, 0.21, P = 0.29) (Figure 5), with no heterogeneity and 28 ± 26 wk of resistance exercise, respectively (gait speed
present. SMD: 0.13; 95% CI: −0.03, 0.28, P = 0.10; chair-rise SMD:
−0.01; 95% CI: −0.16, 0.17, P = 0.95), with no heterogeneity
present (Figure 8A, B).
Muscle cross-sectional area
Protein supplementation on top of resistance exercise was not
associated with greater changes in thigh muscle cross-sectional Publication bias and sensitivity analyses
area (SMD: 0.09; 95% CI: −0.23, 0.42, P = 0.57) compared Considerable symmetry was observed on examining the
with the control condition after 16 ± 6 wk with no heterogeneity individual funnel plots of SE by Hedge’s g and the “trim and
present (Figure 6). fill algorithm,” which implied there was no publication bias
for the analyses (Supplemental Figures 1–7). The “remove-
one” analyses revealed that no single study significantly alters
Muscle strength the magnitude and direction for the analyses of lean body
Protein supplementation for 30 ± 31 wk did not yield greater mass, muscle strength, and physical performance in the analyses
improvement of upper body strength during resistance exercise assessing the effect of protein supplementation compared with
training than the control condition receiving solely resistance controls and in the analyses that assessed the effect of protein
exercise training (SMD: 0.11; 95% CI: −0.07, 0.29, P = 0.23) supplementation compared with controls during concomitant
(Figure 7A), with no heterogeneity present. No significant resistance exercise training.
differences for lower extremity muscle strength were found
between protein supplementation and control conditions after
24 ± 24 wk of resistance exercise training with no hetero- DISCUSSION
geneity present (Figure 7B; SMD: 0.10; 95% CI: −0.06, 0.27, The present work is the first meta-analysis to assess the effect
P = 0.22). of protein supplementation on lean body mass, muscle strength,

Arnarson, 2013 (36) 17.7 -0.15 (-0.48, 0.18) 0.36
Bell, 2017 (34) 6.2 -0.18 (-0.74, 0.38) 0.53

Bemben, 2010, Carter, 2005, Eliot, 2008 (37, 40, 45) 2.6 0.38 (-0.48, 1.25) 0.38
Campbell, 1994, Campbell, 1995 (38, 39) 1.5 0.07 (-1.06, 1.21) 0.90
Candow, 2006 Protein after exercise (20) 2.4 0.05 (-0.85, 0.95) 0.91
Candow, 2006 Protein before exercise (20) 2.5 0.02 (-0.86, 0.90) 0.96
Daly, 2014, Torres, 2017 (43, 65) 11.1 0.50 (0.08, 0.91) 0.02
Holm, 2008 (48) 3.6 0.07 (-0.66, 0.80) 0.85
Iglay, 2007, Iglay, 2009 (49, 50) 4.4 0.07 (-0.60, 0.73) 0.84
Kukuljan, 2009 (33) 10.9 0.14 (-0.28, 0.56) 0.51
Leenders, 2013 Females (54) 3.0 0.04 (-0.76, 0.84) 0.92
Leenders, 2013 Males (54) 3.7 0.07 (-0.66, 0.80) 0.85

Meredith, 1992 (56) 1.4 0.16 (-1.03, 1.35) 0.80
Rossato, 2017 (61) 2.9 0.01 (-0.81, 0.83) 0.98
Seino, 2018 (63) 9.8 0.13 (-0.32, 0.57) 0.57
Thomson, 2016 Dairy (64) 6.9 0.02 (-0.51, 0.55) 0.94
Thomson, 2016 Soy (64) 6.2 0.06 (-0.50, 0.62) 0.84
Verdijk, 2009 (21) 3.3 0.02 (-0.75, 0.79) 0.96

Overall 0.08 (-0.06, 0.21) 0.29
-3.00 -1.50 0.00 1.50 3.00
FIGURE 5 Forest plots of the SMD of protein supplementation during resistance exercise training compared with a control condition during resistance
exercise training on total lean body mass. For each trial, the black square represents the point estimate of the intervention effect. The horizontal line joins the
lower and upper limits of the 95% CI of this effect. The area of the black square represents the relative weight of the study in the meta-analysis. The black
diamond represents the pooled estimate result (SMD) of the random-effects meta-analysis. No heterogeneity was present (Cochran’s Q = 7.32; I2 = 0.0%,
P = 0.98). SMD, standardized mean difference.
and physical performance in nonfrail community-dwelling older supplementation compared with the control condition during
adults. We found no beneficial effects of protein supplementation concomitant resistance exercise training. These observations are
on lean body mass, muscle strength, and chair-rise ability. We contradictory to previously reported outcomes, and suggest that
did find a tendency of a larger increase in handgrip strength protein supplementation may only exert beneficial effects in
and gait speed after protein supplementation compared with the specific groups of older adults.
control condition. In addition, the effect of additional protein
supplementation during resistance exercise training on lean
body mass, muscle thigh cross-sectional area, muscle strength, Effect of protein supplementation
and chair-rise ability was not superior to resistance exercise We found no effects of protein supplementation on lean body
training only in nonfrail older adults, whereas we observed a mass, muscle strength, and/or physical performance in nonfrail
borderline significant larger increase in gait speed with protein community-dwelling older adults, except for a tendency of a

Campbell, 1994, Campbell, 1995 (38, 39) - Midthigh 8.2 0.15 (-0.99, 1.28) 0.80
Godard, 2002 (47) - Midthigh 11.5 0.09 (-0.86, 1.04) 0.85
Holm, 2008 (48) - Mid quadriceps 19.5 0.00 (-0.73, 0.73) 1.00
Leenders, 2013 Females (54) - Quadriceps 16.3 0.05 (-0.75, 0.85) 0.90
Leenders, 2013 Males (54) - Quadriceps 19.7 0.10 (-0.63, 0.83) 0.78
Meredith, 1992 (56) - Midthigh 7.1 0.65 (-0.57, 1.87) 0.30

Verdijk, 2009 (21) - Quadriceps 17.7 -0.02 (-0.79, 0.74) 0.95
Overall 0.09 (-0.23, 0.42) 0.57
-3.00 -1.50 0.00 1.50 3.00
exercise training on thigh muscle cross-sectional area. For each trial, the black square represents the point estimate of the intervention effect. The horizontal
line joins the lower and upper limits of the 95% CI of this effect. The area of the black square represents the relative weight of the study in the meta-analysis.
The black diamond represents the pooled estimate result (SMD) of the random-effects meta-analysis. No heterogeneity was present (Cochran’s Q = 0.97;
I2 = 0.0%, P = 0.99). SMD, standardized mean difference.

A
Bell, 2017 (34) - Total upper body 1RM 10.3 0.13 (-0.43, 0.69) 0.66
Bemben, 2010, Carter, 2005, Eliot, 2008 (37, 40, 45) - Lat pull down 4.4 0.05 (-0.81, 0.91) 0.91
Candow, 2006 Protein after exercise (20) - Bench press 3.9 0.35 (-0.56, 1.26) 0.45
Candow, 2006 Protein before exercise (20) - Bench press 4.2 0.16 (-0.72, 1.04) 0.72
Farnfield, 2012 (46) - Bench press 3.8 -0.03 (-0.96, 0.89) 0.94
Iglay, 2008, Iglay, 2009 (49, 50) - Chest press 6.5 -0.09 (-0.79, 0.62) 0.81
Kukuljan, 2009 (33) - Lat pull down 18.3 0.07 (-0.35, 0.49) 0.75
Mitchell, 2015 (58) - Chest press 3.3 0.41 (-0.58, 1.40) 0.42
Nabuco, 2018 Protein after exercise (59) - Chest press 9.2 0.11 (-0.48, 0.71) 0.71
Nabuco, 2018 Protein before exercise (59) - Chest press 9.5 0.11 (-0.47, 0.70) 0.70
Rossato, 2017 (61) - Bench press 4.8 -0.06 (-0.88, 0.75) 0.88
Thomson, 2016 Dairy (64) - Lat pull down 11.5 0.22 (-0.31, 0.75) 0.41

Thomson, 2016 Soy (64) - Lat pull down 10.3 0.11 (-0.45, 0.67) 0.71
Overall 0.11 (-0.07, 0.29) 0.23
-3.00 -1.50 0.00 1.50 3.00

B
Arnarson, 2013 (36) - Leg extension 12.3 0.05 (-0.28, 0.38) 0.78
Bell, 2017 (34) - Total lower body 1RM 6.5 0.02 (-0.54, 0.58) 0.95
Bemben, 2010, Carter, 2005, Eliot, 2008 (37, 40, 45) - Leg press 2.3 1.99 (0.95, 3.04) <0.01
Candow, 2006 Protein after exercise (20) - Leg press 3.0 0.01 (-0.89, 0.91) 0.98
Candow, 2006 Protein before exercise (20) - Leg press 3.1 0.03 (-0.85, 0.91) 0.94
Daly, 2014, Torres, 2017 (43, 65) - Leg extension 9.7 0.26 (-0.15, 0.67) 0.22
Farnfield, 2012 (46) - Leg press 2.4 1.34 (0.32, 2.36) 0.01
Godard, 2002 (47) - Leg extension 2.7 0.25 (-0.71, 1.20) 0.61
Iglay, 2008, Iglay, 2009 (49, 50) - Leg press 4.6 0.19 (-0.50, 0.89) 0.59
Kukuljan, 2009 (33) - Leg press 9.5 0.02 (-0.41, 0.44) 0.94
Leenders, 2013 (54) - Leg press 6.8 0.02 (-0.52, 0.56) 0.95
Meredith, 1992 (56) - Leg extension 1.8 0.11 (-1.08, 1.29) 0.86
Mitchell, 2015 (58) - Leg press 2.6 -0.28 (-1.27, 0.70) 0.57
Nabuco, 2018 Protein after exercise (59) - Leg extension 6.0 0.00 (-0.59, 0.59) 1.00
Nabuco, 2018 Protein before exercise (59) - Leg extension 6.1 0.00 (-0.58, 0.58) 1.00
Rossato, 2017 (61) - Leg extension 3.5 0.15 (-0.67, 0.97) 0.71
Thomson, 2016Dairy(64) - Leg press
Thomson, 2016 Soy (64) - Leg press 6.3 -0.40 (-0.97, 0.17) 0.17
Verdijk, 2009 (21) - Leg press 7.0 3.9 0.06 (-0.71, 0.83) 0.88
Overall 0.10 (-0.06, 0.27) 0.22
-3.00 -1.50 0.00 1.50 3.00
exercise training on upper body (A) and lower extremity (B) muscle strength. For each trial, the black square represents the point estimate of the intervention
effect. The horizontal line joins the lower and upper limits of the 95% CI of this effect. The area of the black square represents the relative weight of the study
in the meta-analysis. The black diamond represents the pooled estimate result (SMD) of the random-effects meta-analysis. No heterogeneity was present in
both analyses (Cochran’s Q = 1.43, I2 = 0.0%, P = 1.00 and Cochran’s Q = 23.49; I2 = 23.4%, P = 0.17, respectively). RM, repetition maximum; SMD,
standardized mean difference.
larger increase in handgrip strength and gait speed after protein amino acids. Thus, the findings of Scognamiglio et al. (62) should
supplementation compared with the control condition. These be interpreted with care, because they may not be representative
borderline significant findings, however, were hampered by of the effects of protein intake in general nonfrail community-
significant heterogeneity, which was caused by the pronounced dwelling older adults.
positive findings of a single study (62). Participants of this study The absent beneficial effects of protein supplementation in our
in healthy elderly had baseline grip strength and gait speed meta-analysis are in contrast with RCTs that found a positive
scores far below recommendations (68), which allowed for large effect of protein supplementation in frail older adults (69–73).
improvements in handgrip strength and gait speed. Furthermore, It has been suggested that the beneficial effects of protein
participants received a very high dose of supplementation: 35 g supplementation may be different between frail and nonfrail older
A
Arnarson, 2013 (36) - 6 min walk 21.3 -0.08 (-0.41, 0.25) 0.63
Bell, 2017 (34) - 6 min walk 5.7 0.16 (-0.48, 0.79) 0.63
Daly, 2014 (43) - 4 square steptest 13.8 0.05 (-0.36, 0.46) 0.80
Kawada, 2013 3 g amino acids (52) - 6 min walk 2.7 0.16 (-0.77, 1.09) 0.74
Kawada, 2013 6 g amino acids (52) - 6 min walk 2.7 0.52 (-0.40, 1.45) 0.27
Kukuljan, 2009 (33) - 6 m walk test 13.2 0.06 (-0.36, 0.48) 0.80
Nabuco, 2018 Protein after exercise (59) - 10 m walk test 6.4 0.55 (-0.05, 1.15) 0.07
Nabuco, 2018 Protein before exercise (59) - 10 m walk test 6.7 0.47 (-0.13, 1.06) 0.12
Seino, 2018 (63) - 5 m walk test 11.8 0.06 (-0.36, 0.48) 0.80
Thomson, 2016 Dairy (64) - 6 min walk 8.2 0.39 (-0.14, 0.93) 0.15
Thomson, 2016 Soy (64) - 6 min walk 7.4 0.13 (-0.43, 0.69) 0.65
Overall 0.13 (-0.03, 0.28) 0.10

-3.00 -1.50 0.00 1.50 3.00
Study Favors control Favors protein Relative weight SMD (95% CI) P value
B
Arnarson, 2013 (36) - TUG 24.5 -0.07 (-0.40, 0.26) 0.66
Bell, 2017 (34) - TUG 6.6 0.24 (-0.40, 0.88) 0.46

Daly, 2014 (43) - TUG 15.8 -0.04 (-0.45, 0.37) 0.85
Kukuljan, 2009 (33) - 15s steptest 15.2 -0.11 (-0.53, 0.31) 0.60
Leenders, 2013 (54) - 30s sit-to-stand 9.1 -0.01 (-0.56, 0.53) 0.96
Nabuco, 2018 Protein after exercise (59) - 5 times sit-to-stand 7.6 0.29 (-0.30, 0.89) 0.34
Nabuco, 2018 Protein before exercise (59) - 5 times sit-to-stand 7.8 0.31 (-0.28, 0.90) 0.30
Seino, 2018 (63) - TUG 13.6 -0.10 (-0.55, 0.34) 0.65

Overall 0.01 (-0.16, 0.17) 0.95
-3.00 -1.50 0.00 1.50 3.00
exercise training on gait speed (A) and chair-rise performance (B). For each trial, the black square represents the point estimate of the intervention effect. The
horizontal line joins the lower and upper limits of the 95% CI of this effect. The area of the black square represents the relative weight of the study in the
meta-analysis. The black diamond represents the pooled estimate result (SMD) of the random-effects meta-analysis. No heterogeneity was present in both
analyses (Cochran’s Q = 7.16; I2 = 0.0%, P = 0.71 and Cochran’s Q = 3.24; I2 = 0.0%, P = 0.86, respectively). SMD, standardized mean difference; TUG,
Timed Up-and-Go test.
adults (74), because a higher chronic inflammatory activity in with younger individuals (78), especially because postprandial
frail elderly may cause an attenuated muscle protein metabolism muscle protein synthesis is blunted in older adults (79, 80).
compared with nonfrail older adults (8, 75, 76). Therefore, Incorporating doses of 25–30 g protein in the diet of older
protein supplementation may only exert beneficial adaptations in adults has been suggested as a promising strategy to counteract
frail older adults. the attenuated postprandial muscle protein synthesis (81, 82).
There are several other explanations for the lack of ben- Detailed information on protein supplementation protocols was
eficial effects of additional protein on muscle characteristics not provided in most of the studies, so we cannot exclude
in the population of nonfrail community-dwelling older adults the possibility that suboptimal interventions may have affected
presented here. First, the mean habitual protein intake of outcomes of our meta-analysis.
study participants was higher than the Recommended Dietary
Allowance of 0.8 g · kg−1 · d−1 . Therefore, protein intake
might have already been sufficient to counteract the age-related Effect of protein supplementation during concomitant
anabolic resistance (77) and additional protein supplementation resistance exercise training
beyond the Recommended Dietary Allowance may have no Studies have shown that resistance exercise induces a
additional effect on body and muscle characteristics. Given the muscle protein synthetic response in the ageing muscle (83–
curvilinear and saturable dose-response relation between protein 85). Therefore, resistance exercise in combination with protein
intake and muscle protein synthesis (8), it may be possible that supplementation has been proposed to have synergistic effects
nonfrail older individuals reach a plateau phase when a protein on muscle characteristics in older adults (8, 86). We found no
intake >0.8 g · kg−1 · d−1 is achieved. Second, the protein superior effect of protein supplementation in combination with
supplementation protocol could have been suboptimal in some resistance exercise training compared with resistance exercise
studies. Older individuals require higher per-meal protein doses training only on changes in lean body mass, thigh muscle
to achieve similar rates of muscle protein synthesis compared cross-sectional area, muscle strength, and chair-rise ability in
nonfrail older adults. Gait speed was borderline significantly included in our meta-analysis. However, it also induces more
more improved with protein supplementation compared with the heterogeneity, which represents a potential limitation when inter-
control condition during concomitant resistance exercise training. preting the results of this meta-analysis. Significant heterogeneity
These observations align with meta-analyses that assessed the was present in only 2 forest plots, assessing the effect of protein
effects of protein supplementation during concomitant resistance supplementation compared with control conditions on handgrip
exercise training on muscle strength (14, 87), but are contra- strength and gait speed. To correct for possible heterogeneity we
dictory to changes found in lean body mass (14, 88, 89). This adopted a random-effect approach and our sensitivity analyses
apparent discrepancy in lean body mass between our analysis and indicated that our results showed good robustness. Nevertheless,
previous meta-analyses (14, 88, 89) might be partly explained the small number of studies with similar protein supplementation
by the inclusion of studies with (pre)frail participants (18, 19, protocols highlights the need for additional long-term studies that
90) in these meta-analyses. The RCTs in those meta-analyses assess which amount, type, or timing of protein gives beneficial
had a relatively large weight owing to the large sample size effects in a homogeneous population of nonfrail older adults to
of (pre)frail older adults and found a positive effect of protein address whether protein supplementation can delay the onset of
supplementation (14, 88, 89). An alternative explanation for sarcopenia.

the discrepancy may be the very strong correlation coefficient
of r = 0.98 used (88), whereas we used a correlation factor
of r = 0.50 according to reported literature (29, 30). Indeed, Conclusions
our sensitivity analyses on our pooled dataset revealed that In conclusion, protein supplementation in nonfrail
a significant beneficial effect of protein supplementation was community-dwelling older adults does not lead to increases
achieved with a correlation factor r ≥ 0.98, but not with lower in lean body mass, thigh muscle cross-sectional area, muscle
correlation factors. A third explanation for the contradictory strength, or physical performance compared with control
findings of protein intake on lean body mass changes may conditions, nor does it exert superior effects when added
relate to the contribution of protein intake–induced change in to resistance exercise training. Habitual protein intakes of
lean organ mass. Animal studies have demonstrated hypertrophy most study participants were already sufficient and protein
of organs such as the kidney and liver in response to high- interventions differed in relation to type of protein, amount, and
protein diets (91, 92), which may contribute to the increased timing. Future research should focus on optimization of protein
lean body mass. However, the increase in organ mass only intake in nonfrail community-dwelling older adults with low
occurred with high protein levels (92). It is still unclear how habitual protein intake and assess whether these individuals
these findings translate to humans because the hypertrophic could benefit from particular protein supplementation
effects of protein intake on human organs are currently unknown. protocols.
Future studies assessing the distinct effects of protein intake on
muscle mass compared with organ mass are therefore warranted. We thank On Ying Chan who assisted with our extensive literature search
and Maroeska M Rovers for her advice on the analysis.
On the other hand, contradicting results might also be caused
The authors’ responsibilities were as follows—DSMtH, TMHE, and
by differences in protein supplementation protocols between MTEH: designed the research; DSMtH and MAHN: conducted the research;
the RCTs. In our meta-analysis, there are distinct differences DSMtH and MFHM: analyzed the data; DSMtH, AMHH, TMHE, and
among RCTs in the timing of protein intake, and the type MTEH: wrote the manuscript; DSMtH, TMHE, and MTEH: had primary
and amount of protein, but these factors are known to affect responsibility for final content. None of the authors reported a conflict of
changes in body composition and/or muscle characteristics (93). interest related to the study.
Finally, habitual physical activity levels of participants are
often not taken into account, yet these could influence the
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Effects of protein supplementation on lean body mass, muscle

strength, and physical performance in nonfrail community-dwelling

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ABSTRACT Keywords: sarcopenia, elderly, protein, lean body mass, perfor-

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Inclusion criteria Exclusion criteria

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Reporting External validity Confounding Total score

Habitual protein Protein and placebo supplementation

Con 12 (12/0) 70.8 ± 3.5 25.1 ± 2.5 6 1.3 ± 0.2 — — NR N BB

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Prot2 64 (29/35) 61.7 ± 8.3 27.5 ± 3.7 12 1.08 ± 0.09 Soy 27 — — AE

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Aleman-Mateo, 2014 (35) 17.9 0.06 (-0.34, 0.45) 0.77

Chanet, 2017 (42) 4.4 0.10 (-0.70, 0.90) 0.80

Dillon, 2009 (44) 2.5 0.19 (-0.86, 1.24) 0.72

Kerstetter, 2015 (53) 22.1 0.08 (-0.28, 0.44) 0.66

Kukuljan, 2009 (33) 16.2 0.05 (-0.37, 0.48) 0.80

Markofski, 2018 (55) 4.4 0.05 (-0.37, 0.48) 0.80

Mitchell, 2017 (57) 5.2 0.27 (-0.47, 1.00) 0.48

Norton, 2016 (60) 11.0 0.01 (-0.50, 0.51) 0.98

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-3.00 -1.50 0.00 1.50 3.00

Favors control Favors protein

A Study Relative weight SMD (95% CI) P value

Aleman-Mateo, 2014 (35) - Handgrip 14.2 0.08 (-0.32, 0.47) 0.70

-3.00 -1.50 0.00 1.50 3.00

Favors control Favors protein

B Study Relative weight SMD (95% CI) P value

Overall 0.03 (-0.20, 0.27) 0.78

-3.00 -1.50 0.00 1.50 3.00

Favors control Favors protein

A Study Relative weight SMD (95% CI) P value

-3.00 -1.50 0.00 1.50 3.00

Favors control Favors protein

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Overall 0.10 (-0.08, 0.28) 0.26

-3.00 -1.50 0.00 1.50 3.00

Favors control Favors protein

Study Relative weight SMD (95% CI) P value

Bell, 2017 (34) 6.2 -0.18 (-0.74, 0.38) 0.53

Leenders, 2013 Males (54) 3.7 0.07 (-0.66, 0.80) 0.85

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Thomson, 2016 Soy (64) 6.2 0.06 (-0.50, 0.62) 0.84

Verdijk, 2009 (21) 3.3 0.02 (-0.75, 0.79) 0.96

-3.00 -1.50 0.00 1.50 3.00

Favors control Favors protein

Study Relative weight SMD (95% CI) P value

Meredith, 1992 (56) - Midthigh 7.1 0.65 (-0.57, 1.87) 0.30

-3.00 -1.50 0.00 1.50 3.00

Favors control Favors protein

Study Relative weight SMD (95% CI) P value

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Overall 0.11 (-0.07, 0.29) 0.23

-3.00 -1.50 0.00 1.50 3.00

Favors control Favors protein