Selection and identification of studies
Out of 1139 publications that were identified in our initial search, 378 duplicate articles were excluded. After screening the remaining 761 records, 751 unrelated articles were removed based on title and abstract assessment. Then, ten publications remained for further evaluation of the full text. Two eligible articles were published on the same dataset (19, 23, 34), of which the complete one was included (34). In total, nine eligible RCTs were included in our final analysis (18, 20, 23, 34–39). out of them, five studies assessed changes in lumbar-BMD (18, 20, 23, 36, 37). 3 studies hip-BMD (36, 37, 39), 3 NTx (18, 20, 34), 3 studies CTx (35, 37, 38), 3 studies serum Osteocalcin (18, 20, 34) and 2 studies IGF (37, 39). The flow diagram of study selection is outlined in Figur 1.
Characteristics of the included studies:
The characteristics of 9 RCTs included in the current systematic review and meta-analysis are illustrated in Table 2. These RCTs were published between 2001 and 2022. two studies were exclusively performed on male subjects (36, 38), five studies on females (18, 20, 23, 34, 39), and others on both genders (37, 40). the total sample size included 620 individuals. Out of which 319 subjects were in the intervention group, and 301 belonged to the control group The age range of the participants was 18 to 85 years. According to Cochrane scores, seven studies were classified as high-quality studies (more than four items of low risk), and two were classified as moderate-quality (2–3 items of low risk). The result of the quality assessment is reported in Supplementary file 1.
Table 2
Characteristics of the RCTs included in the current systematic review and meta-analysis.
Study | Participants | Country | Mean age | BMI | No. (intervention/ control) | Durations (week) | Intervention | Control | Exercise intervention | Result |
Aoe et al. 2005 | menopausal women (unhealthy) | Japan | 50.5 ± 3.0 | INT: 21.7 ± 2.6 CON: 21.4 ± 3.2 | 27 14/13 | 24 WK | MBP/ 40 mg per day | 50- ml placebo beverage | --- | BMD-lumbar\(\uparrow\) Lumbar-BMD\(\uparrow\) NTx\(\downarrow\) Osteocalcin \(\leftrightarrow\) |
Aoe et al. 2001 | healthy women | Japan | 28.8 ± 8.7 | NR | 33 17/16 | 24 WK | MBP/ 40 mg per day | 50- ml placebo beverage | --- | NTx\(\downarrow\) Osteocalcin\(\uparrow\) |
Uenishi et al. 2006 | healthy young women | Japan | 21.3 ± 1.2 | INT: 21.0 ± 2.3 CON: 20.7 ± 2.3 | 35 17/18 | 24 WK | MBP/ 40 mg per day | 50-ml placebo beverage | --- | BMD-lumbar\(\uparrow\) NTx\(\downarrow\) Osteocalcin\(\uparrow\) |
Zou et al. 2009 | healthy young women | China | 19.6 ± 0.6 | INT: 20.7 ± 1.7 CON: 20.5 ± 2.2 | 53 29/24 | 32 WK | MBP /250 ml whole milk added with 40 mg MBP | nothing | --- | BMD-lumbar\(\uparrow\) BMD %\(\uparrow\) |
Fuglsang-Nielsen et al. 2022 | Adult with abdominal obesity (both) | Denmark | ≥ 40 | INT: 28 ± 4 CON: 30 ± 4 INT: 29 ± 2 CON: 29 ± 4 | 31 15/16 16/17 | 12 WK | 60 g/d whey hydrolysate + 10g/d fiber Or 60 g/d whey hydrolysate + 30g/d fiber | 60 g/d maltodextrin + 10g/d fiber Or 60 g/d maltodextrin + 30g/d fiber | 3d per week Resistance training | CTx\(\leftrightarrow\) |
Kerstetter et al. 2015 | healthy Men and women | USA | 69.95 ± 6.25 | INT: 26.1 ± 3.4 CON: 26.1 ± 3.4 | 92/79 | 36 OR 72 WK | 45 g whey protein isolate | maltodextrin | --- | BMD-hip \(\leftrightarrow\) BMD-lumbar \(\leftrightarrow\) CTx\(\uparrow\) IGF\(\uparrow\) |
kemler et al. 2020 | osteosarcopenic men unhealthy | Germany | ≥ 72 years | NR | 21/22 | 72 WK | 100 g protein powder (360 kcal) contained 80 g of (whey) protein | nothing | twice/week high intensity dynamic resistance exercise | BMD-hip\(\uparrow\) BMD-lumbar\(\uparrow\) |
Sefton et al. 2020 | Healthy male soldiers | USA | 22.10 ± 3.51 | NR | 23/25 | 9 WK | 38.6 g of protein consisting of 80% whey protein concentrate | carbohydrate in liquid-shake | physical training | CTx\(\leftrightarrow\) |
Zhu et al. 2011 | healthy ambulant women, | Australia | 70–80 | INT: 26.1 ± 3.8 CON: 27.2 ± 4.0 | 179 91/88 | 12 WK | 30 g of whey protein isolate | placebo drink containing 2.1 g of protein | ---- | BMD-hip\(\downarrow\) IGF\(\uparrow\) |
Abbreviations. BMI: body mass index, CON: control group, INT: intervention group, WK: week, MBP: milk basic protein, BMD: bone mineral density, NTx: N-terminal telopeptide of type I collagen, NR: non-report, CTx: C-terminal telopeptide of type I collagen |
Findings from the systematic review:
Among seven studies assessing the effect of whey or MBP supplementation on BMD, four studies revealed a significant increasing effect (18, 20, 23), 2 found no significant effect (36, 37), and one found decreasing substantial effect (39). Of these, four studies related to MBP supplements (18, 20, 23, 34) and three related to whey supplements (36, 37, 39). Three studies assessed NTx changes (18, 20, 34), which all reported significant NTx reductions. Three studies assessed CTx changes (35, 37, 38), of which one reported a significant increase (37) and 2 described no changes (35, 38). Related to changes in Osteocalcin, two trials illustrated significant increases following MBP supplementation (18, 34) while one did not (20), and two trials reported serum IGF, which was found effective.
Findings from the meta-analysis
Overall, 9 RCTs in the systematic review were included in the meta-analysis. These trials had a total sample size of 620 individuals aged 18 years and over.
The effect of whey and MBP on Lumbar-BMD:
Based on the results of 6 effect sizes (5 studies) (18, 20, 23, 36, 37), we did not find a significant effect of whey and MBP supplementation on lumbar-BMD [weighted mean difference (SMD): -0.08 g/c\({m}^{2}\), 95CI: -0.33 to 0.16, P = 0.529, Fig. 2a]. Heterogeneity between the studies was low (\({I}^{2}\) = 35.9%, p = 0.167).
Sensitivity analysis showed that the overall effect size regarding the effects of whey and MBP supplementation on lumbar-BMD levels did not depend on a single study (CI range: -0.33, 0.16).
Subgroup analyses by type of supplementation
Based on the results of six effect sizes, three arms from whey (36, 37) and three from BMD (18, 20, 23), whey or MBP supplementation had no significant effect on lumbar-BMD (p = 0.367 and p = 0.859, respectively, Table 3).
Table 3
lumbar BMD |
Subcategories | Effect size, n | I2(%) | P-heterogeneity | SMD | (95%CI) | P-value |
Type | | | | | | |
Whey | 3 | 64.8 | 0.058 | -0.17 | -0.54 to 0.20 | 0.368 |
MBP | 3 | 0.0 | 0.402 | 0.03 | -0.33 to 0.40 | 0.859 |
Pooled | 6 | 35.9 | 0.167 | -0.08 | -0.33 to 0.16 | 0.491 |
durations | | | | | | |
≤ 36wk | 4 | 0.0 | 0.606 | 0.01 | -0.22 to 0.25 | 0.910 |
> 36 wk | 2 | 80.6 | 0.023 | -0.35 | -1.13 to 0.43 | 0.376 |
Hip BMD |
Type | | | | | | |
whey | 4 | 96.3 | 0.001 | -0.23 | -1.17 to 0.70 | 0.626 |
durations | | | | | | |
≤ 36 wk | 2 | 98.1 | 0.001 | -0.83 | -2.46 to 0.80 | 0.318 |
> 36 wk | 2 | 81.1 | 0.021 | 0.36 | -0.43 to 1.15 | 0.374 |
NTx |
Type | | | | | | |
MBP | 6 | 84.6 | 0.001 | -0.89 | -1.69 to -0.10 | 0.028 |
durations | | | | | | |
≤ 12 wk | 3 | 91.8 | 0.001 | -0.78 | -2.35 to -0.79 | 0.331 |
> 12 wk | 3 | 65.5 | 0.055 | -1.02 | -1.76 to -0.27 | 0.007 |
CTx |
Type | | | | | | |
whey | 5 | 98.6 | 0.001 | 2.10 | -0.55 to 4.75 | 0.121 |
Exercising condition | | | | | | |
exercise | 3 | 0.0 | 0.780 | -0.22 | -0.59 to 0.15 | 0.247 |
No exercise | 2 | 0.0 | 0.353 | 5.59 | 5.03 to 6.16 | 0.001 |
Osteocalcin |
Type | | | | | | |
MBP | 6 | 93.9 | 0.001 | 0.39 | -0.95 to 1.73 | 0.576 |
durations | | | | | | |
≤ 12 wk | 3 | 95.7 | 0.001 | -0.33 | -2.60 to 1.94 | 0.774 |
> 12 wk | 3 | 93.8 | 0.001 | 1.10 | 0.78 to 2.99 | 0.251 |
IGF |
Type | | | | | | |
whey | 3 | 58.1 | 0.092 | 3.55 | 3.12 to 3.98 | 0.001 |
Abbreviation: SMD: standard mean difference, CI: confidence interval, MBP: milk basic protein, wk: week, |
Subgroup analyses by type of duration
Based on the results of six effect sizes, four arms from ≤ 36week (18, 20, 23, 37) and two from > 36 (36, 37), whey and MBP supplementation had no significant effect on lumbar-BMD among duration ≤ 36 weeks or > 36 weeks (p = 0.910 and p = 0.376, respectively, Table 3).
The effect of whey on Hip-BMD:
Pooled effect size from 3 studies (36, 37, 39) containing four arms did not reveal a significant change in Hip-BMD following whey protein supplementation [SMD: -0.23 g/c\({m}^{2}\), 95% CI: -1.17 to 0.70%, P = 0.626, Fig. 2b]. Heterogeneity between the studies was high (\({I}^{2}\) = 96.3%, p = 0.001). Sensitivity analysis showed that the overall effect size regarding the effects of whey supplementation on hip-BMD levels did not depend on a single study (CI range: -1.17, 0.70).
Subgroup analyses by type of duration
Based on the results of 4 effect sizes (36, 37, 39), two arms from ≤ 36week (37, 39) and two from > 36 (36, 37), whey and MBP supplementation had no significant effect on hip-BMD among duration ≤ 36 weeks or > 36 weeks (p = 0.318 and p = 0.378, respectively, Table 3).
The effect of MBP on NTx:
In total, six effect sizes from 3 RCTs (18, 20, 34) were included in the meta-analysis. Combining the effect sizes, these studies show large heterogeneity among effect sizes (I2 = 84.6%, P = 0.001). MBP supplementation induced significant decrease in NTx [SMD: -0.89 nmol/mmol, CI: -1.69 to -0.10%, P = 0.028, Fig. 2c]. Sensitivity analysis showed that the overall effect size regarding the effects of MBP supplementation on NTx levels depends on a single study (Aoe et al. 2001 (34)) (CI range: -1.21, 0.07).
Subgroup analyses by type of duration
Based on the results of six effect sizes, three arms from ≤ 12week and three from > 12, MBP supplementation had no significant effect on NTX among duration ≤ 12 (p = 331, Table 3) but had a considerable decrease effect on NTx among duration > 12 weeks (p = 0.007, Table 3).
The effect of whey and whey protein on CTx:
The combined effect size of 3 studies (35, 37, 38) containing five arms demonstrate no significant change in CTx following whey protein supplementation [SMD: 2.1 ng/l, 95% CI: -0.55 to 4.75%, P = 0.121, Fig. 2d]. These studies show large heterogeneity among effect sizes (I2 = 98.6%, P = 0.001). Sensitivity analysis showed that the overall effect size regarding the effects of whey supplementation on CTx levels depends on no single study (CI range: -0.55 to 4.75).
Subgroup analyses by exercising condition
Based on the five effect sizes, whey supplementation (three arms) had no significant effect on CTx among exercising conditions (p = 0.247, Table 3). The pooled effect of why supplementation (two arms) on CTx among no exercising conditions was found the increase to be significant (p = 0.001, Table 3).
The effect of whey and MBP on osteocalcin: based on the result of 3 studies (18, 20, 34) containing 6 effect size, MBP supplementation failed to change osteocalcin [SMD: 0.39 ng/ml, 95% CI: -0.95 to 1.73%, P = 0.576, Fig. 2e]. Heterogeneity between the studies was high (\({I}^{2}\) = 93.9%, p = 0.001). Sensitivity analysis showed that the overall effect size regarding the effects of MBP supplementation on Osteocalcin did not depend on a single study (CI range: -0.95, 1.73).
Subgroup analyses by type of duration
Based on the results of six effect sizes, three arms from ≤ 12week (18, 20, 34) and three from > 12 (18, 20, 34), MBP supplementation had no significant effect on Osteocalcin among duration ≤ 12 weeks or > 12 weeks (p = 0.774 and p = 0.251, respectively, Table 3).
The effect of whey supplementation on IGF:
After combining three effect sizes from 2 studies (37, 39), pooled effects data analysis indicated that whey supplementation resulted in a significant increase in IGF [SMD: 3.55 nmol/l, 95% CI: 3.12 to 3.98%, P = 0.001, \({I}^{2}\) = 58.1%, p = 0.092, Fig. 2f]. Sensitivity analysis showed that the overall effect size regarding whey supplementation's effects on IGF levels depend on no study (CI range: 3.12, 3.98 ).
Publication bias
According to Begg regression test, there was no evidence of publication bias for studies examining the effect of whey and MBP supplementation on lumbar-BMD (p = 0.851 ), hip-BMD (p = 1.000), osteocalcin (p = 0.707) and IGF (p = 0.296). in addition, Eggers regression test showed no significant publication bias for lumbar-BMD (p = 0.415), hip-BMD (p = 0.763), NTx (p = 0.032), CTx (p = 0.121), osteocalcin (p = 0.856) and IGF (p = 0.090).but a publication bias was found for NTx (Begg, p = 0.024 and egger, p = 0.032) and CTx (Begg,p = 0.027). we did the trim-and-fill method and found that adding one missing studies made no effect on significant of NTX and CTx (SMD: -1.09 nmol/mmol, 95% CI: -1.89, -2.69 and SMD: 1.15 ng/l ,95% CI: -1.51, 3.83, respectively) Fig. 3 (A_F).