nutrients

Article
Effect of Whey Protein Supplementation in Postmenopausal
Women: A Systematic Review and Meta-Analysis
Yao-Yi Kuo 1,2,† , Hao-Yun Chang 1,3,† , Yu-Chen Huang 4,5,6 and Che-Wei Liu 4,7,8,9, *

1 School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan
2 Division of General Medicine, Department of Medical Education, Shuang Ho Hospital,
Taipei Medical University, New Taipei City 235, Taiwan
3 Division of General Medicine, Department of Medical Education, Far Eastern Memorial Hospital,
New Taipei City 220, Taiwan
4 Research Center of Big Data and Meta-Analysis, Wan Fang Hospital, Taipei Medical University,
Taipei 116, Taiwan
5 Department of Dermatology, Wan Fang Hospital, Taipei Medical University, Taipei 116, Taiwan
6 Department of Dermatology, School of Medicine, College of Medicine, Taipei Medical University,
Taipei 110, Taiwan
7 Department of Orthopedics, Cathay General Hospital, Taipei 106, Taiwan
8 School of Medicine, College of Medicine, Fu Jen Catholic University, New Taipei City 242, Taiwan
9 School of Medicine, National Tsing Hua University, Hsinchu 300, Taiwan
* Correspondence: petergates38@[Link]; Tel.: +886-983701350
† These authors contributed equally to this work.

Abstract: (1) Background: Whey protein (WP) in combination with resistance training (RT) is benefi-
cial in improving sarcopenic obesity and its damaging effects in older adults, while the difference
between men and women should be considered while interpreting results. This review aims to inves-
tigate WP’s efficacy on postmenopausal women with or without RT; (2) Material and Methods: We
searched electronic databases including PubMed, EMBASE, and the Cochrane Library from inception
to August 2021 for randomized controlled trials that included comparison groups to evaluate WP’s
Citation: Kuo, Y.-Y.; Chang, H.-Y.;
Huang, Y.-C.; Liu, C.-W. Effect of
efficacy in women aged 55 years and above. The outcomes included body composition, muscular
Whey Protein Supplementation in strength, functional capacity, and dietary intake. Standardized mean differences (SMDs) with 95%
Postmenopausal Women: A confidence intervals (CIs) were used to estimate the effect of WP. We also performed subgroup analy-
Systematic Review and Meta- sis with or without RT; (3) Results: We included 14 studies in the systematic review and 10 studies
Analysis. Nutrients 2022, 14, 4210. in the meta-analysis. Subgroup analyses showed RT was a major confounder for muscle strength,
[Link] lean mass, and dietary protein intake (PI). In the RT subgroup, WP supplementation had a significant
Academic Editor: Monica Serra
positive effect on biceps curl strength (BC) (SMD: 0.6805, 95% CI: 0.176, 1.185, I2 : 0%), and lower
limb lean-mass (LLLM) (SMD: 1.103, 95% CI: 0.632, 1.574, I2 : 14%). In the subgroup without RT, a
Received: 16 August 2022 significant negative effect on PI (SMD: −0.4225, 95% CI: −0.774, −0.071, I2 : 47%) was observed, while
Accepted: 7 October 2022
no significant effect on muscle strength or lean mass was revealed. WP supplementation did not show
Published: 10 October 2022
a significantly different effect on fat mass or body weight loss in both the subgroups; (4) Conclusions:
Publisher’s Note: MDPI stays neutral In postmenopausal women, WP supplementation only in combination with RT enhances BC and
with regard to jurisdictional claims in LLLM compared to placebo controls. Without RT, WP has no significant benefit on muscle strength
published maps and institutional affil- or lean mass.
iations.

Keywords: postmenopausal; resistance training; sarcopenia; whey protein; women

Copyright: © 2022 by the authors.

Licensee MDPI, Basel, Switzerland.
1. Introduction
This article is an open access article
distributed under the terms and Aging is an irreversible process which predisposes individuals to many biological
conditions of the Creative Commons alterations, often associated with a progressively decreased muscle mass and strength
Attribution (CC BY) license (https:// (sarcopenia), and an increased fat mass (obesity) [1]. This combination, termed sarcopenic
[Link]/licenses/by/ obesity, can cause chronic inflammation thus explaining the increased risk of developing
4.0/). metabolic syndrome and comorbidities, such as type-2 diabetes mellitus, dyslipidemia,

Nutrients 2022, 14, 4210. [Link] [Link]

Nutrients 2022, 14, 4210 2 of 16

and cardiovascular diseases [2,3]. Moreover, the reduction in muscular function capacity
enhances the risk of falls and fractures. This reduces physical performance and causes
higher mortality in older individuals [4,5]. Thus, measures to attenuate the age-related
health declines and to improve the quality of life of the elderly have become a global effort.
Older women generally possess lower levels of muscle mass and muscular strength,
and consume a lower baseline protein intake (PI) as compared to men [6,7]. The decrease
in physical function may also contribute to a higher risk of falls. Moreover, older post-
menopausal women have increased total cholesterol and LDL-c, decreased HDL-c, elevated
blood pressure, and insulin resistance due to a lack of estrogen protection [8]. Hence,
older women with these risks should be considered susceptible to the damaging effects
of sarcopenia.
Whey protein (WP) is commonly used as protein supplementation compared to other
sources of protein as it is easily digested and leads to rapid amino acid delivery to skeletal
muscles, making it easier to meet the body’s protein requirements. It is rich in leucine and
is therefore, considered more effective in stimulating muscle protein synthesis [9,10].
A previous prospective cohort study indicated that higher dairy PI has lesser association
with low muscle mass and abdominal obesity [11]. Previous studies comparing WP and an
ordinary protein-rich diet via dietary counseling demonstrated that if the amount of protein
reaches a threshold (1.2–1.5 g/kg BM−1 day−1), muscle mass can be increased in sarcopenic
elders. Additionally, under achieving adequate protein intake (1.2–1.5 g/kg BM−1 day−1), WP
can further improve gait speed compared to the ordinary protein-rich diet group in the
sarcopenic elderly [12].
The combination of resistance training (RT) and protein supplementation are the
most common non-pharmacological strategies for older adults to attenuate age-related
changes in metabolic and body composition [13]. RT provides mechanical stimuli that
are considered effective for increasing muscle mass and strength, reducing fat mass and
inflammatory biomarkers, and improving metabolic syndrome profile [14]. Moreover,
functional training which uses movements similar to performing activities of daily living
also improves neuromuscular qualities, e.g., balance, flexibility, and endurance, resulting
in a better quality of life for the elderly [15].
The effects of protein with or without RT may differ according to the population
characteristics, such as the age group, gender, and the protein chosen. The results may be
confounded by the proportion of both genders included, as the differences in metabolism
between men and women should be considered [16,17]. Previous studies have assessed
the additive effect of higher protein consumption on RT, which showed an increase in
muscle mass, strength, and improved performance in the elderly compared to control
group [18–21].
WP has already demonstrated its potential role in RT within the general population.
However, there is currently no consensus on the effects of protein supplementation on
muscular adaptions and nutritional intake in older women. This systematic review and
meta-analysis with randomized controlled trials (RCTs) was conducted to investigate the
efficacy of WP and evaluate the differences between elderly women with or without RT.

2. Materials and Methods

This study was conducted in accordance with the 2020 Preferred Reporting Items for
Systematic Reviews and Meta-Analyses guidelines (Table S1 (Supplementary Materials)),
and was registered on PROSPERO (CRD42022330726).

2.1. Literature Search Strategy

Relevant studies were identified from the following electronic databases: PubMed,
EMBASE, and the Cochrane Library from inception to August 2021 without any language
restriction. The primary search terms in the title and abstract were used as follows: (whey
protein) AND (menopause women) OR (older women)) for selecting eligible studies. The
detailed search strings are shown in Method S1. To broaden the search scope, additional
Nutrients 2022, 14, 4210 3 of 16

studies were obtained by screening the citations of relevant studies from “similar articles”
listed in PubMed.

2.2. Eligibility Criteria and Study Selection

The eligible studies should meet the following inclusion criteria: (1) A randomized
controlled trial that included comparison groups to evaluate the efficacy of diet supple-
mented with WP, either with or without RT; (2) Studies that used validated tools to evaluate
body composition, lean mass (depicted as lean mass, muscle mass, or lean soft tissue
mass), muscular strength, functional capacity, and dietary intake outcomes; and (3) Study
participants that included women above 55 years of age. We excluded articles that met at
least one of the following criteria: (1) Study types other than RCTs; (2) Non-human research;
and (3) Insufficient outcome data. Titles and abstracts of potentially eligible studies were
screened, their full texts were retrieved, and reviewed based on the eligibility criteria. Two
independent authors (H. Y. Chang and Y. Y. Kuo) were involved in the retrieval, screening
and selection process.

2.3. Data Extraction and Quality Assessment

Two independent authors (H. Y. Chang and Y. Y. Kuo) extracted the data from the
included studies. The extracted data included the author’s surname, year of publication,
country, ethnicity, sample size, participant characteristics, exercise interventions, changes
between baseline and endpoint on study outcomes, study duration, and time points of
data collection. The methodological quality of each included study was assessed by using
the modified Cochrane Risk of Bias tool Version 2.0, which included the following five
domains: allocation bias, performance bias, attrition bias, detection bias, and reporting bias.
The overall bias of each article was determined to be low, unclear, or high risk. A third
reviewer (Y.C. Huang) was consulted for any disagreement to reach a consensus.

2.4. Data Synthesis and Statistical Analysis

The primary outcomes were changes in muscular strength and functional capacity, and
gain of lean mass; the secondary outcomes were changes in fat mass, loss of body weight,
and dietary intake from baseline study outcome to the treatment endpoint. Meta-analyses
were performed when at least two studies compared the same outcome in participants
receiving or not receiving WP supplementation. A subgroup analysis for participants with
or without RT was also performed. Continuous variables were expressed by standardized
mean differences (SMDs) with 95% confidence intervals (CIs). A random-effects model was
adopted due to possible heterogeneity. Data heterogeneity was assessed using the Cochran
Q-test and I2 test. Both funnel plot and Egger’s test were used to assess publication bias
if more than 10 studies were available [22]. All of the analyses were performed using
Comprehensive Meta-Analysis Version 3 software (Biostat, Inc., Englewood, NJ, USA).

2.5. Quality of Evidence

The Grading of Recommendations, Assessment, Development, and Evaluations (GRADE)
approach including risk of bias, inconsistency, indirectness, imprecision, and publication
bias was used to evaluate the quality of evidence of each outcome in this systematic review
and meta-analysis.

3. Results
3.1. Search Results and Trial Characteristics
The primary search terms initially identified 330 records potentially eligible for inclu-
sion. After excluding 109 duplicated records along with 25 records that were reviewed,
guidelines, or conference reports, a total of 196 records entered the screening stage. After
assessing the title or abstract, 140 records were excluded as they were irrelevant to our
research, resulting in a total of 56 reports that were sought for retrieval and assessed for
 3. Results
3.1. Search Results and Trial Characteristics
The primary search terms initially identified 330 records potentially eligible for in-
clusion. After excluding 109 duplicated records along with 25 records that were reviewed,
Nutrients 2022, 14, 4210 guidelines, or conference reports, a total of 196 records entered the screening stage. After4 of 16
assessing the title or abstract, 140 records were excluded as they were irrelevant to our
research, resulting in a total of 56 reports that were sought for retrieval and assessed for
eligibility.
eligibility. Forty-two
Forty-tworeports
reportswere
wereexcluded
excludedas
asthey
theymet
metthe
theexclusion
exclusioncriteria
criteriaor
ordid
didnotnot
meeting
meeting the inclusion criteria. Fourteen reports were included in the review (Figure 1).
the inclusion criteria. Fourteen reports were included in the review (Figure 1).

Figure
[Link]
Flowdiagram.
[Link]
Flowdiagram
diagramof
ofthe
theliterature
literaturesearch
searchand
andselection
selectionprocess.
process.

All14
All 14included
includedstudies
studieswere
wereRCT
RCTwith
withaacontrol
controlgroup.
[Link]
Amongthese,
these,22were
weresingle
single
blinded [23,24], and 12 were double blinded [25–36]. These studies were implemented
blinded [23,24], and 12 were double blinded [25–36]. These studies were implemented in six
in
different countries with the majority having an intervention duration of 12 weeks
six different countries with the majority having an intervention duration of 12 weeks (ranged
from 10 weeks to 72 weeks). Detailed study characteristics are shown in Tables 1 and 2.
(ranged from 10 weeks to 72 weeks). Detailed study characteristics are shown in Tables 1
Among these 14 studies, 11 studies included the data of WP with RT [23–32,35], and
and 2. Among these 14 studies, 11 studies included the data of WP with RT [23–32,35],
4 studies included the data of WP without RT [23,33,34,36]. Furthermore, seven studies were
and 4 studies included the data of WP without RT [23,33,34,36]. Furthermore, seven stud-
conducted by the same team and the participants were from the same group [26–32]. To
ies were conducted by the same team and the participants were from the same group [26–
address the concern of bias due to overlapped participants, we only selected one study with
32]. To address the concern of bias due to overlapped participants, we only selected one
the largest number of participants among these seven studies for each outcome. In addition,
study with the largest number of participants among these seven studies for each
another two studies were conducted by the same team with the same participants [34,36].
However, they did not present the same outcomes and we included both of them in our
study. We reported each study rather than collapsing to one to clarify the difference between
these studies. Finally, four studies were excluded in the meta-analysis for no outcome
selected [26,28,29,32], while 10 studies were included in the meta-analysis for quantitative
analysis [23–25,27,30,31,33–36].
Nutrients 2022, 14, 4210 5 of 16

Table 1. Characteristics of the intervention studies.

Number of Female Outcome

Study, Year Intervention Resistance Whey Protein Placebo Supple-
Female Participant Measured and
Country Duration Training Supplementation mentation
Participants Health Status Used
3 days/week
3 sets × 8–12 reps CP
20 g × 2/day maltodextrin
Ashley et al., 2 upper limb GST GS
12 weeks With RT: 67 Well-fed healthy post RT or before non-isocaloric
2021 [25] USA exercises BW
meal non-nitrogenous
1 lower limb exercise FM
1 aerobic exercise
2 days/week
2 sets to fatigue
4 upper limb
Kirk et al., 2020 With RT: 25 Non-frail healthy 1.5 g/kg BM−1 SMM KF GS KE
16 weeks exercises -
[23] UK Without RT: 27 RT naïve day−1 FM
3 lower limb
exercises
moderate weight
3 days/week
3 sets × 8–12 reps
Nabuco et al., Healthy or maltodextrin
4 upper limb 27.1 g
2019, 1st [26] 12 weeks With RT: 44 sarcopenic isocaloric -
exercises post RT
Brazil RT naïve nitrogenous
4 lower limb
exercises
3 days/week
3 sets × 8–12 reps
Nabuco et al., Healthy or maltodextrin
4 upper limb 27.1 g
2019, 2nd [27] 12 weeks With RT: 44 sarcopenic isocaloric FM
exercises post RT
Brazil RT naïve nitrogenous
4 lower limb
exercises
3 days/week
3 sets × 8–12 reps
Nabuco et al., Healthy or maltodextrin
4 upper limb 27.1 g
2019, 3rd [28] 12 weeks With RT: 30 sarcopenic isocaloric -
exercises post RT
Brazil RT naïve nitrogenous
4 lower limb
exercises
3 days/week
3 sets × 8–12 reps
Nabuco et al., maltodextrin
Sarcopenic 4 upper limb 27.1 g
2019, 4th [29] 12 weeks With RT: 26 isocaloric -
RT naïve exercises post RT
Brazil nitrogenous
4 lower limb
exercises
3 days/week
3 sets × 8–12 reps TEI
Healthy or maltodextrin
Sugihara et al., 4 upper limb 27.1 g CHI
12 weeks With RT: 31 sarcopenic isocaloric
2018 [30] Brazil exercises post RT PI
RT naïve nitrogenous
4 lower limb FI
exercises
ULLM
3 days/week SMM
3 sets × 8–12 reps BC
Healthy or maltodextrin
Nabuco et al., 4 upper limb 27.1 g CP
12 weeks With RT: 44 sarcopenic isocaloric
2018 [31] Brazil exercises post RT GST
RT naïve nitrogenous
4 lower limb LLLM
exercises KE
RFSP
2 days/week
ULLM
>40 min
GST
2 upper limb
Mori et al., 2018 Healthy 22.3 g LLLM
24 weeks With RT: 50 exercises -
[24] Japan RT naïve post RT GS
5 lower limb
KE
exercises 50–70%
BW
1RM
3 days/week
3 sets × 8–12 reps
Healthy or maltodextrin
Fernandes et al., 4 upper limb 27.1 g
12 weeks With RT: 32 sarcopenic isocaloric -
2018 [32] Brazil exercises post RT
RT naïve nitrogenous
4 lower limb
exercises
Nutrients 2022, 14, 4210 6 of 16

Table 1. Cont.

Number of Female Outcome

Study, Year Intervention Resistance Whey Protein Placebo Supple-
Female Participant Measured and
Country Duration Training Supplementation mentation
Participants Health Status Used
FM
TEI
Stojkovic et al., maltodextrin
72 weeks Without RT: 84 - - 20 g CHI
2017 [33] USA isocaloric
PI
FI
ULLM
SMM
KF
carbohydrate
Zhu et al., 2015 30 g (milk and LLLM
52 weeks Without RT: 196 - - isocaloric
[34] Australia whey protein) GS
nitrogenous
KE
BW
RFSP
2 days/week
0–6 sets to fatigue
Weisgarber maltodextrin BC
Healthy 2 upper limb
et al., 2014 [35] 10 weeks With RT: 12 40 g isocaloric KF
RT naïve exercises
Canada non-nitrogenous KE
2 lower limb
exercises 30% 1 RM
TEI
Hodgson et al., carbohydrate
30 g (milk and CHI
2012 [36] 52 weeks Without RT: 196 - - isocaloric
whey protein) PI
Australia nitrogenous
FI
BC = biceps curl strength; BW = body weight; CHI = carbohydrate intake; CP = chest press; FI = fat intake;
FM = fat mass; GS = grip strength; GST = gait speed test; KE = knee extension strength; KF = knee flexion strength;
LLLM = lower limb lean mass; PI = protein intake; PLA = placebo; reps = repetitions; RT = resistance training;
RFSP = rising from sitting position; SMM = skeletal muscle mass; TEI = total energy intake; ULLM = upper limb
lean mass; WP = whey protein.

Table 2. Participant characteristics of the intervention studies.

Relative Protein
Study, Year Age BMI (kg·m−2 ) FM (kg) KE (kg) GS (kg) Intake
(g/kg BM−1 Day−1 )
26.851 ± 0.80
23.639 ± 1.37 (WP)
Ashley et al., 2021 61.93 ± 1.23 (WP) (WP) 1.1 ± 0.09 (WP)
- 24.25 ± 0.89 -
[25] 60.64 ± 0.93 (PLA) 27.88 ± 0.75 1.20 ± 0.09 (PLA)
(PLA)
(PLA)
Nabuco et al., 2018,
66.2 ± 9.4 (WP) 25.3 ± 5.4 (WP) 23.2 ± 8.4 (WP) 55.0 ± 11.0 (WP) 0.94 ± 0.34 (WP)
2019 1st, 2nd -
66.5 ± 7.1 (PLA) 23.8 ± 3.7 (PLA) 22.9 ± 7.5 (PLA) 52.0 ± 13.0 (PLA) 0.95 ± 0.27 (PLA)
[26,27,31]
Nabuco et al., 2019,
68.0 ± 4.2 (WP) 26.4 ± 3.0 (WP) 23.8 ± 5.4 (WP) 48.7 ± 10.8 (WP) 0.93 ± 0.36 (WP)
4th -
70.1 ± 3.9 (PLA) 27.4 ± 3.0 (PLA) 23.8 ± 5.9 (PLA) 50.9 ± 9.9 (PLA) 0.97 ± 0.28 (PLA)
[29]
Mori et al., 2018 70.6 ± 4.2 (WP) 22.1 ± 2.1 (WP) 23.8 ± 6.3 (WP) 22.4 ± 3.4 (WP) 1.3 ± 0.0 (WP)
-
[24] 70.6 ± 4.2 (control) 22.9 ± 2.9 (control) 26.7 ± 3.8 (control) 23.1 ± 5.3 (control) 1.3 ± 0.0 (control)
Stojkovic et al., 2017 68.9 ± 0.9 (WP) 26.0 ± 0.6 (WP) 25.9 ± 1.3 (WP)
- - -
[33] 69.3 ± 0.9 (PLA) 25.8 ± 0.6 (PLA) 25.5 ± 1.1 (PLA)
Weisgarber et al.,
59.7 ± 15.3 (WP)
2014 57 ± 4.7 28.3 ± 7.0 - - -
61.0 ± 16.1 (PLA)
[35]
173 ± 46 (MVC) 23.2 ± 5.5 (MVC)
Kirk et al., 2020 (RT) 69 ± 6 (WP) 27.4 ± 4.9 (WP) 22.8 ± 10.5 (WP) (WP) (WP) 1.16 ± 0.4 (WP)
[23] 66 ± 4 (control) 28.1 ± 7.4 (control) 28.2 ± 17.6 (control) 233 ± 126 (MVC) 21.7 ± 4.8 (MVC) 1.10 ± 0.4 (control)
(control) (control)
190 ± 105 (MVC) 22.4 ± 4.4 (MVC)
Kirk et al., 2020
72 ± 6 (WP) 27.1 ± 4.1 (WP) 27.6 ± 8.5 (WP) (WP) (WP) 0.99 ± 0.2 (WP)
(non-RT)
68 ± 6 (control) 26.2 ± 4.5 (control) 25.5 ± 11.9 (control) 180 ± 49 (MVC) 23.9 ± 4.1 (MVC) 0.98 ± 0.3 (control)
[23]
(control) (control)
Nutrients 2022, 14, 4210 7 of 16

Table 2. Cont.

Relative Protein
Study, Year Age BMI (kg·m−2 ) FM (kg) KE (kg) GS (kg) Intake
(g/kg BM−1 Day−1 )
Nabuco et al., 2019, 69.2 ± 4.1 (WP) 27.4 ± 5.1 (WP) 0.94 ± 0.30 (WP)
- - -
3rd [28] 68.4 ± 4.5 (PLA) 26.6 ± 3.4 (PLA) 0.96 ± 0.22 (PLA)
Sugihara et al., 2018 67.4 ± 4.1 (WP) 25.6 ± 2.4 (WP) 25.7 ± 4.6 (WP) 52.7 ± 10.3 (WP) 0.85 ± 0.1 (WP)
-
[30] 67.8 ± 4.1 (PLA) 25.4 ± 2.6 (PLA) 26.2 ± 5.8 (PLA) 52.8 ± 13.3 (PLA) 0.81 ± 0.1 (PLA)
Fernandes et al.,
67.3 ± 4.1 (WP) 25.9 ± 2.7 (WP) 25.7 ± 4.6 (WP) 52.7 ± 10.3 (WP) 0.85 ± 0.1 (WP)
2018 -
67.8 ± 4.0 (PLA) 25.4 ± 2.6 (PLA) 26.2 ± 5.8 (PLA) 52.8 ± 13.3 (PLA) 0.81 ± 0.1 (PLA)
[32]
Zhu et al., 2015 74.2 ± 2.8 (WP) 26.1 ± 3.8 (WP) 15.4 ± 5.3 (WP) 21.7 ± 5.2 (WP) 1.2 ± 0.3 (WP)
-
[34] 74.3 ± 2.6 (PLA) 27.2 ± 4.0 (PLA) 16.1 ± 7.2 (PLA) 21.7 ± 5.5 (PLA) 1.1 ± 0.3 (PLA)
Hodgson et al., 2012 74.2 ± 2.8 (WP) 26.1 ± 3.8 (WP) 1.1 ± 0.3 (WP)
- - -
[36] 74.3 ± 2.6 (PLA) 27.2 ± 4.0 (PLA) 1.1 ± 0.3 (PLA)
FM = fat mass; KE = knee extension strength; GS = grip strength; MVC= maximum voluntary contraction;
PLA = placebo; RT = resistance training; WP = whey protein.

3.2. Risk of Bias Assessment

The risk of bias assessment of the studies included in this systematic review is pre-
sented in Figure 2a,b. High risk of bias was only seen in the domain of attrition bias in one
study for incomplete outcome data [35]. In that study, 12 participants completed the study
while the outcome data of biceps curl strength (BC), knee extension strength, and knee
flexion strength were available from only 10 of them. There was no evidence that the result
was unbiased, and the missing data likely depended on true value. Therefore, attrition bias
in this study was rated as high.

3.3. Participants’ Characteristics

The total participants across the included studies were 776, with average age ranging
from 57 to 74 years and average BMI ranging from 22 to 28 kg/m2 . Participants were
declared to be “healthy” in four studies [23–25,35], and to be “healthy or sarcopenic” in three
studies [27,30,31]. Participants were classified to be PI deficient in two studies, reporting a
PI at baseline lower than the current adult recommended dietary-allowances [30,32].

3.4. RT Characteristics
Among the 10 included studies, 7 studies included the data of WP with RT. The RT
interventions duration ranged from 10 to 24 weeks undergoing RT for 2 to 3 days per week,
with 4–8 exercises per session, with 0–6 sets per exercise, and with 8–12 repetitions per set
(or to fatigue). All the studies involved upper and lower body RT. Each outcome of muscle
strength was trained by specific RT programs in all the studies.

3.5. WP Characteristics
Among the 10 included studies, 9 studies orally gave a range of 20 to 40 g of daily protein
supplement [24,25,27,30,31,33–36]. Eight studies used WP [23–25,27,30,31,33,35], and two
studies used skimmed milk plus WP isolate [34,36]. Furthermore, six studies provided protein
supplements after RT in addition to regular meals on no RT days [24,25,27,30,31,35]. Four
studies provided protein supplements daily in addition to regular meals [23,33,34,36]. All con-
trol groups did not receive protein supplementation. Eight studies used placebo supplements
mainly consisting of carbohydrates with similar flavor [25,27,30,31,33–36]. Among them,
seven studies used placebo supplements with similar calories content [27,30,31,33–36].
Two studies did not use placebo supplements [23,24].
 - - -
[36] 74.3 ± 2.6 (PLA) 27.2 ± 4.0 (PLA) 1.1 ± 0.3 (PLA)
FM = fat mass; KE = knee extension strength; GS = grip strength; MVC= maximum voluntary con-
traction; PLA = placebo; RT = resistance training; WP = whey protein.

Nutrients 2022, 14, 4210

3.2. Risk of Bias Assessment 8 of 16

(a)

(b)
Figure
Figure2.2.(a).
(a).Risk
Riskof
of bias
bias assessment. Presentedasasa apercentage
assessment. Presented percentage across
across allall included
included studies;
studies; (b). (b).
RiskRisk
ofofbias
biasassessment.
assessment. For all included
For all includedstudies
studies[24–36].
[24–36].

3.3.
[Link]’ Characteristics
Results of Meta-Analysis
The detailed data of each included study are shown in Tables S2–S5. The results of the
meta-analysis are shown in Table 3.
Nutrients 2022, 14, 4210 9 of 16

Table 3. The results and GRADE evaluation of all outcomes.

Combined WP with RT and without RT

Mean Difference,
Outcome (PLA vs. Number of Quality of
with 95% Confidence I2 Risk of Bias Inconsistency Indirectness Imprecision
WP) Articles Evidence
Interval
Upper limb lean −0.2415 (−0.473,
3 1.5% −1 −0 −0 −0 Moderate
mass −0.01)
Lower limb lean
3 0.028 (−0.214, 0.27) 93.6% −1 −1 −0 −0 Low
mass
Skeletal muscle
3 0.08 (−0.533, 0.693) 67% −1 −1 −0 −0 Low
mass
Grip strength 5 0.1005 (−0.107, 0.308) 0% −1 −0 −0 −0 Moderate
Biceps curl
2 0.6805 (0.176, 1.185) 0% −2 −0 −0 −0 Low
strength
Knee extension
6 0.027 (−0.194, 0.248) 44% −2 −0 −0 −0 Low
strength
Knee flexion
4 0.044 (−0.214, 0.302) 22% −2 −0 −0 −0 Low
strength
Gait speed test 3 0.1625 (−0.261, 0.586) 44% −1 −0 −0 −1 Low
Rising from −0.036 (−0.291,
2 0% −1 −0 −0 −0 Moderate
sitting position 0.219)
Chest press 2 0.108 (−0.268, 0.484) 0% −1 −0 −0 −1 Low
−0.107 (−0.443,
Fat mass 5 93% −1 −1 −0 −1 Very low
0.229)
−0.115 (−0.291,
Body weight 4 0% −1 −0 −0 −0 Moderate
0.061)
−0.0685 (−0.396,
Protein intake 3 94% −0 −1 −0 −1 Low
0.259)
−0.085 (−0.311,
Fat intake 3 0% −0 −0 −0 −0 High
0.141)
Carbohydrate −0.0205 (−0.365,
3 32% −0 −0 −0 −1 Moderate
intake 0.324)
Total energy −0.067 (−0.293,
3 2.84% −0 −0 −0 −0 High
intake 0.159)
WP with RT
Upper limb lean
2 0 (−0.405, 0.405) 0% −1 −0 −0 −1 Low
mass
Lower limb lean
2 1.103 (0.632, 1.574) 14% −1 −0 −0 −0 Moderate
mass
Skeletal muscle
2 0.4775 (−0.473, 1.428) 72% −1 −0 −0 −1 Low
mass
Grip strength 3 0.1765 (−0.156, 0.509) 0% −1 −0 −0 −0 Moderate
Biceps curl
2 0.6805 (0.176, 1.185) 0% −2 −0 −0 −0 Low
strength
Knee extension
4 0.364 (−0.031, 0.759) 24% −2 −0 −0 −1 Very low
strength
Knee flexion
2 0.2905 (−0.756, 1.337) 67% −2 −1 −0 −1 Very low
strength
Gait speed test 3 0.1625 (−0.261, 0.586) 44% −1 −0 −0 −1 Low
Chest press 2 0.108 (−0.268, 0.484) 0% −1 −0 −0 −1 Low
−0.0885 (−0.427,
Fat mass 3 0% −1 −0 −0 −1 Low
0.25)
−0.0205 (−0.386,
Body weight 2 0% −1 −0 −0 −1 Low
0.345)
WP without RT
Grip strength 2 0.0515 (−0.215, 0.318) 0% −1 −0 −0 −0 Moderate
Knee extension −0.1255 (−0.392,
2 0% −1 −0 −0 −0 Moderate
strength 0.141)
Knee flexion
2 0.0285 (−0.238, 0.295) 0% −1 −0 −0 −0 Moderate
strength
Nutrients 2022, 14, 4210 10 of 16

Table 3. Cont.

Combined WP with RT and without RT

Mean Difference,
Outcome (PLA vs. Number of Quality of
with 95% Confidence I2 Risk of Bias Inconsistency Indirectness Imprecision
WP) Articles Evidence
Interval
−1.2825 (−3.975,
Fat mass 2 96% −1 −1 −0 −1 Very low
1.410)
−0.1435 (−0.344,
Body weight 2 0% −1 −0 −0 −0 Moderate
0.057)
−0.4225 (−0.774,
Protein intake 2 47% −0 −1 −0 −0 Moderate
−0.071)
−0.1135 (−0.352,
Fat intake 2 0% −0 −0 −0 −0 High
0.125)
Carbohydrate
2 0.038 (−0.356, 0.432) 58% −0 −0 −0 −1 Moderate
intake
Total energy −0.1225 (−0.361,
2 0% −0 −0 −0 −0 High
intake 0.116)
PLA = placebo; RT = resistance training; WP = whey protein.

3.7. Effect of WP Supplementation on Muscle Strength and Functional Capacity

We included knee flexion strength (KF), grip strength (GS), knee extension strength
(KE), chest press (CP), gait speed test (GST), biceps curl strength (BC), and rising from
sitting position (RFSP) in the muscle strength and functional capacity assessment. The
participants with WP supplementation showed no significant difference in the change of
KF (SMD: 0.044, 95% CI: −0.214, 0.302,), GS (SMD: 0.101, 95% CI: −0.107, 0.308,), KE (SMD:
0.027, 95% CI: −0.194, 0.248), and RFSP (SMD: −0.036, 95% CI: −0.291, 0.219), compared to
those without WP supplementation. (Figures S1–S7)
In the RT subgroup, the pooled estimate revealed, the participants receiving WP had a
significant increase in BC (SMD: 0.6805, 95% CI: 0.176, 1.185) than those without WP. There
was no difference in the change of KF (SMD: 0.2905, 95% CI: −0.756, 1.337), GS (SMD:
0.1765, 95% CI: −0.156, 0.509), KE (SMD: 0.364, 95% CI: −0.031, 0.759), CP (SMD: 0.108,
95% CI: −0.268, 0.484), and GST (SMD: 0.1625, 95% CI: −0.261, 0.586).
Meta-analyses of CP, BC, and GST were not performed in the WP group without
RT due to lack of data availability. The results of WP group without RT revealed that
participants receiving WP showed no significant change of KF (SMD: 0.0285, 95% CI:
−0.238, 0.295), GS (SMD: 0.0515, 95% CI: −0.215, 0.318,), and KE (SMD: −0.1255, 95% CI:
−0.392, 0.141).

3.8. Effect of WP Supplementation on Muscle Mass Gains

We included upper-limb lean mass (ULLM), lower-limb lean mass (LLLM), and skele-
tal muscle mass (SMM) in the muscle mass gain-assessment. The results showed that WP
supplementation may not enhance the outcomes related to muscle mass. A significant
negative effect was demonstrated in ULLM (SMD: 0.2415, 95% CI: −0.473, −0.01), while
that of LLLM (SMD: 0.028, 95% CI: −0.214, 0.27) and SMM (SMD: 0.080, 95% CI: −0.533,
0.693) did not show a significant difference (Figures S8–S10).
In the RT subgroup, WP supplementation may enhance all of the outcomes related
to muscle mass except for ULLM, which showed no effect. Moreover, a significant effect
was demonstrated only in LLLM (SMD: 1.103, 95% CI: 0.632, 1.574), while no significant
effect was shown in ULLM (SMD: 0, 95% CI: −0.405, 0.405,), and SMM (SMD: 0.4775, 95%
CI: −0.473, 1.428).
Meta-analyses of ULLM, LLLM and SMM were not performed in the WP group
without RT due to the lack of data availability.

3.9. Effect of WP Supplementation on Fat Mass (FM) and Body Weight Loss (BW)
The participants with WP supplementation showed an increase in FM and BW loss rel-
ative to the PLA groups, while no significant effect was demonstrated, including FM
Nutrients 2022, 14, 4210 11 of 16

(SMD: −0.107,95% CI: −0.443, 0.229) and BW (SMD: −0.115, 95% CI: −0.291, 0.061).
(Figures S11 and S12).
In the RT subgroup, the pooled estimate revealed that participants receiving WP
showed an increase in FM loss (SMD: −0.0885, 95% CI: −0.427, 0.25), and BW loss (SMD:
−0.0205, 95% CI: −0.386, 0.345) relative to the PLA groups without significant difference.
In the subgroup of participants without RT, the results revealed that WP supple-
mentation showed an increase in FM loss (SMD: −1.2825, 95% CI: −3.975, 1.410), and
BW loss (SMD: −0.1435, 95% CI: −0.344, 0.057) relative to the PLA groups without
significant difference.

3.10. Effect of WP Supplementation on Daily Dietary Nutrients Intake

We regarded daily dietary intake as an outcome in our study to investigate how the
introduction of a daily supplement impacted on daily macronutrient and energy intakes.
We included total energy intake (TEI), carbohydrate intake (CHI), protein intake (PI), and
fat intake (FI) in daily dietary nutrient intake assessment. We used the dietary nutri-
ent intake with deduction of the WP and placebo supplementation to render the results
more objective. Two studies presented total nutrient intake including WP and placebo
supplementation [30,36], while they both provided the nutrient composition of the supple-
ments. Therefore, the dietary nutrient intake with WP and the placebo supplementation
deduction were calculated according to the supplementation protocol.
The participants with WP supplementation showed that WP supplementation may
reduce PI (SMD: −0.0685, 95% CI: −0.396, 0.259), TEI (SMD: −0.067, 95% CI: −0.293, 0.159),
CHI (SMD: −0.0205, 95% CI: −0.365, 0.324) and FI (SMD: −0.085, 95% CI: −0.311, 0.141)
without significant difference. No result was presented in the subgroup analysis of WP
with RT as there was only one study available for each outcome. (Figures S13–S16)
In the subgroup without RT, WP supplementation may enhance CHI and reduce PI,
FI, and TEI. A significant effect was demonstrated in PI (SMD: −0.4225, 95% CI: −0.774,
−0.071), while that of CHI (SMD: 0.038, 95% CI: −0.356, 0.432), FI (SMD: −0.1135, 95% CI:
−0.352, 0.125), and TEI (SMD: −0.1225, 95% CI: −0.361, 0.116) were not significant.

3.11. Quality of Evidence Assessment by GRADE

The results of the GRADE evaluation are presented in Table 3. We evaluated all of the
outcomes with more than one paper providing information. Therefore, all 17 outcomes were
assessed in the analysis of combining all included studies, while only 11 and 10 outcomes
were assessed in the subgroup analysis of WP supplementation with and without RT,
respectively. As the design of the inclusion criteria was rigid, there was no obvious
intransitivity. In the analysis of combining all of the included studies, the confidence of
the evidence of FI and TEI were high for no downgrading, while that of FM was very low
due to the inconsistency, imprecision, and some concerns in relation to the risk of bias. In
the subgroup assessment, the extent of inconsistency decreased, while that of imprecision
increased. For the subgroup of WP supplementation with RT, the confidence of the evidence
of KE and KF was very low due to the imprecision and high risk in relation to the risk of
bias. For the subgroup analysis of WP supplementation without RT, the confidence of the
evidence of FM was very low due to the inconsistency, imprecision, and some concerns in
relation to the risk of bias.

4. Discussion
Based on evidence, this review summarized the effect of WP supplementation on
postmenopausal women. The main findings of this meta-analysis are: (1) WP with RT
significantly enhances LLLM gain and BC; (2) WP without RT significantly reduces PI.

4.1. Effect of WP Supplementation on Muscle Strength and Functional Capacity

Subgroup analysis showed that RT augments the benefits of whey protein for muscle
strength. WP supplementation in the group with RT demonstrated a significant enhance-
Nutrients 2022, 14, 4210 12 of 16

ment of BC, while that without RT showed no significant enhancement of all kinds of
muscle strength and functional capacity. The analysis results partially agree with previous
meta-analyses by Finger et al., Morton et al., and Liao et al. [19,20,37], which investigated
the effect of protein supplementation together with RT, and a previous meta-analysis by
Richard et al., suggesting that protein interventions augment the effect of RT on muscle
strength in older adults. Therefore, despite the difference in metabolism between men and
women, for postmenopausal women consuming sufficient quantities of protein, WP sup-
plementation still enhances muscle strength, but only when combined with RT. A possible
explanation is that RT increases fasted-state protein losses and the need for protein. In this
condition, WP supplementation promotes maintaining whole body protein-balance [38].
Another study concluded that the improvement in strength was related to better
physical and social function [39]. Nevertheless, in our study, no significant effect on
functional capacity was revealed. More studies with uniformity in outcome measures
would be needed to clarify this conclusion.

4.2. Effect of WP Supplementation on Muscle Mass

RT played an important role for muscle mass in the subgroup analysis. WP supple-
mentation in the RT group showed a significant enhancement in LLLM. However, the
group without RT demonstrated a significant decrease in ULLM and LLLM. The results of
our analysis partially agree with previous meta-analyses [19–21,37], suggesting that protein
interventions augment the effect of RT on appendicular lean mass. For the significant
decrease in ULLM and LLLM, the results were constructed by only one study and the
intervention time of this study was one year [34], while that of the studies presenting the
result of WP with RT was 10–24 weeks, and the effect of age-related decline was taken into
consideration. Moreover, this study used an isocaloric carbohydrate for supplementation
in control group. Carbohydrate stimulates pancreatic insulin secretion, which can inhibit
muscle protein breakdown [40,41]. Elderly people of normal status have lower insulin
secretion [42]. This might explain the decrease in ULLM and LLLM.
The results of WP supplementation in the RT group did not show a significant effect
on lower limb muscle strength, but demonstrated a significant enhancement of LLLM. A
previous study presented the same outcome [43], showing that the relationship between
muscle strength and muscle mass differed according to sex and age. For women between
65 and 74 years, no significant relationship between muscle strength and muscle mass was
demonstrated. Both “neural” and “muscular” factors are required for muscle strength;
meanwhile, this neural activity decreases with aging [44]. In addition, strength tests are
performance tests and affected by technique and motivation. In novice individuals, there is
more testing variability. These might explain the lack of a relationship between lower limb
muscle strength and LLLM.

4.3. Effect of WP Supplementation on FM and BW Loss

Subgroup analysis showed that RT was not a key factor in FM and BW loss. Both WP
groups, with and without RT, showed a positive effect on FM loss, corresponding to most
studies showing that increasing dietary protein increases diet-induced thermogenesis and
promotes greater fat loss. Compared to the group of WP without RT, WP supplementation
with RT showed less effect on FM loss. A possible explanation is that the intervention time
of this study was 18 months [33], while that of the other three studies which provided the
results of FM loss were 12–16 weeks [23,24,27]. With a longer intervention time, the effect
on FM loss might be more significant.
Our results demonstrated that WP supplementation might not be necessary to be
combined with RT for FM loss in postmenopausal women.

4.4. Effect of WP Supplementation on Daily Dietary Nutrients Intake

WP supplementation in the group without RT demonstrated a negative effect on
PI without significant difference, while that with RT showed a significant positive ef-
Nutrients 2022, 14, 4210 13 of 16

fect on PI. The results of our analysis partially agree with a previous meta-analysis by
Colonetti et al. [45], which investigated the effect of dietary protein supplementation to-
gether with RT. For the negative effect on PI in the group without RT, it might be explained
by the use of the dietary nutrient intake with deduction of WP and placebo supplementa-
tion in our study. The Study Group on meeting protein needs of older people (PROT-AGE)
and the European Society for Clinical Nutrition and Metabolism (ESPEN) study showed
that 1.0–1.2 g/kg BM−1 day−1 of protein for well-nourished active older adults is suffi-
cient [46,47], while RT can increase fasted-state protein losses and the need of protein. Total
protein intake remained unchanged in the group of WP without RT. With extra supple-
mentation of protein, a new balance of dietary intake was achieved, thus contributing to
a decline in PI. This might indirectly conclude that WP supplementation was not very
helpful for postmenopausal women without RT, especially for those who already consumed
sufficient quantities of protein at baseline. On the contrary, for those who underwent RT,
WP supplementation might augment the effect of RT and increase the need for protein.
Therefore, WP supplementation should be combined with RT for increasing daily dietary
PI in postmenopausal women.

4.5. Limitation
First, due to restricted number of included studies, subgroup analysis other than RT/non-
RT is not constructed in our study. We did not divide the group into trained/untrained
individuals, healthy/sarcopenia individuals, sufficient/deficient PI individuals, or build a
subgroup analysis of protein supplementation dosage. We also did not discriminate the
role of WP from being a supplement or a correction of a deficient diet. Furthermore, some
studies suggested that resistance-trained individuals might need a higher dietary intake and
protein supplementation [20,41]. Second, the intervention time in non-RT studies is longer
than RT studies. The adherence to RT, WP, and PLA supplementation, and the effect of
aging on muscle mass, muscle strength, and FM should be considered. Third, we included
lean mass, muscle mass, and lean soft tissue mass in the analysis of lean mass gain, and the
variation in bone and water should be also considered. Fourth, participants in Sugihara
et al. had a basal PI lower than the current adult-recommended dietary allowances, and
the results of TEI, CHI, PI, and FI should be interpreted with caution. Last, the restricted
number of included studies might lead to some results not showing a significant effect.

5. Conclusions
Compared to placebo control, WP supplementation causes an improvement in BC and
LLLM in postmenopausal women, only when combined with RT. However, the quality of
evidence of BC and LLLM in the group of WP with RT was low and moderate, respectively.
With both of them presenting some risk of bias, the results should be interpreted with
caution. More large scale RCTs are required for a better understanding of the effects of WP
supplementation combined with RT.

Supplementary Materials: The following supporting information can be downloaded at: https:
//[Link]/article/10.3390/nu14194210/s1, Method S1: The full search strings: example of
PubMed; Table S1: PRISMA 2020 checklist; Table S2: Differences in muscle strength between whey
protein supplements and control supplements considering the subgroup analysis of RT/non-RT;
Table S3: Differences in muscle mass between whey protein supplements and control supplements
considering the subgroup analysis of RT/non-RT; Table S4: Differences in fat mass and body weight
between whey protein supplements and control supplements considering the subgroup analysis
of RT/non-RT; Table S5: Differences in daily nutrients intake between whey protein supplements
and control supplements considering the subgroup analysis of RT/non-RT; Figure S1: Forest plot
of the grip strength; Figure S2: Forest plot of the biceps curl strength; Figure S3: Forest plot of the
knee extension strength; Figure S4: Forest plot of the knee flexion strength; Figure S5: Forest plot of
the gait speed test; Figure S6: Forest plot of the chest press; Figure S7: Forest plot of the rising from
sitting position; Figure S8: Forest plot of the upper limb lean mass; Figure S9: Forest plot of the lower
limb lean mass; Figure S10: Forest plot of the skeletal muscle mass; Figure S11: Forest plot of the fat
Nutrients 2022, 14, 4210 14 of 16

mass; Figure S12: Forest plot of the body weight; Figure S13: Forest plot of the total energy intake;
Figure S14: Forest plot of the carbohydrate intake; Figure S15: Forest plot of the fat intake; Figure S16:
Forest plot of the protein intake.
Author Contributions: Y.-Y.K., H.-Y.C., Y.-C.H. and C.-W.L. designed the review; Y.-Y.K. and H.-Y.C.
conducted the systematic review and extracted data; Y.-C.H. performed the data analysis; Y.-Y.K.
and H.-Y.C. wrote the paper; C.-W.L. supervised the project and had primary responsibility for final
content. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Ethical review and approval were waived due to this study
being a systematic review and meta-analysis.
Informed Consent Statement: Patient consent was waived due to this study being a systematic
review and meta-analysis.
Data Availability Statement: Data were available within the article and its supplementary materials.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviation
BC Biceps curl strength
BW Body weight
CHI Carbohydrate intake
CIs Confidence intervals
CP Chest press
FI Fat intake
FM Fat mass
GS Grip strength
GST Gait speed test
KE Knee extension strength
KF Knee flexion strength
LLLM Lower limb lean mass
PI Protein intake
PLA Placebo
RCTs Randomized controlled trials
reps Repetitions
RT Resistance training
RFSP Rising from sitting position
SMM Skeletal muscle mass
SMDs Standardized mean differences
TEI Total energy intake
The Grading of Recommendations,
GRADE
Assessment, Development, and Evaluations
ULLM Upper limb lean mass
WP Whey protein

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