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Dietary protein and bone health: A systematic review and meta-analysis from
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DOI: 10.3945/ajcn.116.145110

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Dietary protein and bone health: a systematic review and meta-analysis

from the National Osteoporosis Foundation1,2
Marissa M Shams-White,3,4 Mei Chung,3 Mengxi Du,3,4 Zhuxuan Fu,3 Karl L Insogna,5 Micaela C Karlsen,4
Meryl S LeBoff,6,7 Sue A Shapses,8 Joachim Sackey,3,4 Taylor C Wallace,9,10* and Connie M Weaver11
3
Department of Public Health and Community Medicine, School of Medicine, and 4Friedman School of Nutrition Science and Policy, Tufts University, Boston,
MA; 5Yale Bone Center at the Yale School of Medicine, Yale University, New Haven, CT; 6Skeletal Health and Osteoporosis Center and Bone Density Unit,

Downloaded from ajcn.nutrition.org at BOSTON UNIVERSITY on January 2, 2018

Harvard Medical School, Boston, MA; 7Endocrine, Diabetes and Hypertension Division, Brigham and Women’s Hospital, Boston, MA; 8Department of
Nutritional Sciences, Rutgers University, New Brunswick, NJ; 9National Osteoporosis Foundation, Arlington, VA; 10Department of Nutrition and Food
Studies, George Mason University, Fairfax, VA; and 11Department of Nutrition Science, Women’s Global Health Institute, Purdue University, West
Lafayette, IN

ABSTRACT
Background: Considerable attention has recently focused on dietary
protein’s role in the mature skeleton, prompted partly by an interest in
nonpharmacologic approaches to maintain skeletal health in adult life.
Objective: The aim was to conduct a systematic review and meta-
analysis evaluating the effects of dietary protein intake alone and
with calcium with or without vitamin D (Ca6D) on bone health
measures in adults.
Design: Searches across 5 databases were conducted through Oc-
tober 2016 including randomized controlled trials (RCTs) and pro-
spective cohort studies examining 1) the effects of “high versus
low” protein intake or 2) dietary protein’s synergistic effect with
Ca6D intake on bone health outcomes. Two investigators indepen-
dently conducted abstract and full-text screenings, data extractions,
and risk of bias (ROB) assessments. Strength of evidence was rated
by group consensus. Random-effects meta-analyses for outcomes
with $4 RCTs were performed.
Results: Sixteen RCTs and 20 prospective cohort studies were
included in the systematic review. Overall ROB was medium.
Moderate evidence suggested that higher protein intake may
have a protective effect on lumbar spine (LS) bone mineral density
(BMD) compared with lower protein intake (net percentage change:
0.52%; 95% CI: 0.06%, 0.97%, I2: 0%; n = 5) but no effect on
total hip (TH), femoral neck (FN), or total body BMD or bone
biomarkers. Limited evidence did not support an effect of protein
with Ca6D on LS BMD, TH BMD, or forearm fractures; there
was insufficient evidence for FN BMD and overall fractures.
Conclusions: Current evidence shows no adverse effects of higher
protein intakes. Although there were positive trends on BMD at most
bone sites, only the LS showed moderate evidence to support bene-
fits of higher protein intake. Studies were heterogeneous, and con-
founding could not be excluded. High-quality, long-term studies are
needed to clarify dietary protein’s role in bone health. This trial was
registered at www.crd.york.ac.uk as CRD42015017751. Am J
Clin Nutr 2017;105:1528–43.
Keywords: protein, bone health, osteoporosis, bone density, diet
INTRODUCTION
Protein makes up w50% of bone volume and approximately
one-third of its mass (1). It provides the structural matrix of
bone, whereas calcium is the dominant mineral within that
matrix. Collagen and a variety of noncollagenous proteins form
the organic matrix of bone, so an adequate dietary protein intake
would seem to be essential for optimal acquisition and main-
tenance of adult bone mass. Consistent with this idea, a recent
systematic review of available data found a positive effect of
dietary protein on skeletal acquisition in children and adoles-
cents (2). Considerable attention has recently also focused on
dietary protein’s role in the mature skeleton, prompted in part
by an increasing interest in nonpharmacologic approaches to
maintaining skeletal health in adult life and later adult years.
In 1920, Sherman (3) reported that increasing dietary protein
led to greater urinary calcium excretion. Subsequent studies have
repeatedly confirmed this association, such that for every 40-g
increase in dietary protein, urinary calcium excretion increases
by w50 mg (4). In the 1970s and early 1980s a series of met-
abolic balance studies in which dietary calcium was carefully
controlled found no change in intestinal calcium absorption with
higher dietary protein intakes, leading to the conclusion that the
source of the additional urinary calcium must be from the break-
down of the skeleton (5–12). It was proposed that increasing di-
etary protein with a commensurate increase in sulfur-containing
1
Supported by an unrestricted educational grant from the National Oste-
oporosis Foundation through the support of the Egg Nutrition Center and
Dairy Management Inc.
2
Supplemental Tables 1–11, Supplemental Methods 1 and 2, Supple-
mental Analytic Data Sets, and Supplemental Figure 1 are available from the
“Online Supporting Material” link in the online posting of the article and
from the same link in the online table of contents at http://ajcn.nutrition.org.
*To whom correspondence should be addressed. E-mail: taylor.wallace@
me.com.
Received September 7, 2016. Accepted for publication March 7, 2017.
First published online April 12, 2017; doi: 10.3945/ajcn.116.145110.

1528 Am J Clin Nutr 2017;105:1528–43. Printed in USA. Ó 2017 American Society for Nutrition

Supplemental Material can be found at:

http://ajcn.nutrition.org/content/suppl/2017/04/12/ajcn.116.1
45110.DCSupplemental.html
 SYSTEMATIC REVIEW OF PROTEIN AND BONE HEALTH
amino acids resulted in the imposition of a systemic metabolic
acid load, which could not—particularly in older individuals—
be adequately buffered by the respiratory apparatus. Instead, this
required buffering from alkali stores in bone resulting in skeletal
calcium loss. However, current short-term dietary intervention
studies that used dual-stable calcium isotopes to assess calcium
kinetics found that increasing dietary protein is associated with a
significant increase in intestinal calcium absorption, such that
nearly the entire increase in urinary calcium can be accounted
for by improved calcium absorption efficiency and that, in the
short term, there is no increase in skeletal catabolism (13–15).
Studies that examined the association between protein intake
and bone health outcomes are limited. A meta-analysis published in
2009 found null effects of protein intake on fracture risk (16);
however, a more recent 2015 meta-analysis indicated a slight re-
duction in hip fractures (17). Moreover, the effects of simultaneous
consumption of protein and calcium intake on bone have not been
widely studied, although a higher protein intake does increase in-
testinal calcium absorption in dual-stable calcium isotope studies
(12–14). Because the body only absorbs limited amounts of these 2
essential nutrients at a time, one could speculate that concurrent
consumption of moderate amounts of protein and calcium at each
meal might offer an advantage. In light of several new studies and a
plethora of available data on bone mineral density (BMD)12, bone
biomarkers, and fracture outcomes, we undertook a comprehensive
systematic review in an effort to clarify the impact of 1) dietary
protein and 2) dietary protein and calcium with or without vitamin
D (Ca6D) on these bone health outcomes in healthy adults.
METHODS
The PRISMA (Preferred Reporting Items for Systematic
Reviews and Meta-Analyses) statement is followed in reporting
this systematic review (18). A prospectively developed proto-
col for this systematic review was registered on PROSPERO
(http://www.crd.york.ac.uk/PROSPERO/display_record.asp?
ID=CRD42015017751; CRD42015017751) (19).
Data sources and searches
A search strategy was developed in consultation with 2 li-
brarians, first by using nomenclatures for Ovid Medline and then
adjusted for other electronic databases. The searches were
implemented in 5 databases: Ovid Medline (gateway.ovid.com;
1946 to 4 October 2016), Cochrane Central Register of Con-
trolled Trials (gateway.ovid.com; 1991 to 31 October 2016),
Scopus (+ EMBASE; www.scopus.com; 1974 to 31 October
2016), Web-of-Science (webofknowledge.com; 1864 to 31 Oc-
tober 2016), and Global Health (1910 to 31 October 2016; www.
ebscohost.com/academic/global-health). The searches were lim-
ited to the English language and human studies that examined the
relations of protein intake (foods or supplement sources) on bone
health outcomes of interest. The complete search strategy is
presented in Supplemental Table 1.
12
Abbreviations used: BMC, bone mineral content; BMD, bone mineral
density; Ca6D, calcium with or without vitamin D; CTX, collagen type 1
cross-linked C-terminal telopeptide; FFQ, food-frequency questionnaire; FN,
femoral neck; LS, lumbar spine; RCT, randomized controlled trial; ROB,
risk of bias; SOE, strength of evidence; TB, total body; TH, total hip.
1529
Study eligibility criteria
We included all intervention trials and prospective cohort studies
in healthy adults aged $18 y that examined the relations between
varying doses of protein intake from any source and fracture and
bone health outcomes. We excluded studies that compared equal
amounts of protein from different protein sources, studies in
children and adolescents (i.e., ,18 y of age), and studies in
pregnant or lactating women. The complete list of bone health
outcomes is described in Table 1, and further details describing the
included study populations and outcome-specific inclusion and
exclusion criteria are described in Supplemental Methods 1.
Study selection process
For citations identified from the non-Medline databases, titles
were first screened by a single investigator to exclude in vitro, cell,
and stem cell studies; animal studies; cross-sectional studies;
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retrospective case-control studies; interrupted time series studies;
and review articles. All of the abstracts identified in the litera-
ture searches were then independently screened by $2 in-
vestigators by using the open-source online software Abstrackr
(http://abstrackr.cebm.brown.edu/) (20). For all abstracts that
were deemed potentially relevant by 2 screeners, full-text articles
were retrieved and independently screened by 2 investigators on
the basis of the final study eligibility criteria. For studies con-
ducted in the same cohort population and time period, the first
published study was retained. All abstract and full-text screening
conflicts were resolved through discussion, and final decisions
were reached by the consensus of the entire research team. Ad-
ditional study eligibility criteria were added after full-text
screening was completed to make the scope of this systematic
review manageable. Specifically for this systematic review, any
study with an intervention duration of ,6 mo was excluded, and
the bone health biomarkers of interest were limited to osteocalcin
and collagen type 1 cross-linked C-terminal telopeptide (CTX) as
measures of bone formation and bone turnover, respectively.
These decisions were made on the basis of their biological and
clinical importance in relation to bone health (e.g., bone density
changes cannot reliably be measured in shorter intervals) (2).
Data extraction
Data extraction forms were tailored to our topic and outcomes of
interest by modifying the data extraction forms used in the sys-
tematic review “Vitamin D and Calcium: A Systematic Review of
Health Outcomes” (21). The items extracted included the fol-
lowing: study characteristics, baseline population characteristics,
background diet data, dietary assessment methods, interventions
(for intervention studies only), confounders and effect modifiers
used in statistical analysis, relevant outcomes assessed, and results
(complete data extraction forms are available on request). Each
study was extracted by one investigator and reviewed and con-
firmed by another investigator. Any disagreements were discussed
among the research team and resolved via group consensus.
Intervention and cohort study results were extracted quanti-
tatively unless such data were not provided; in the latter case,
qualitative results were extracted only. For all studies, multivariate-
adjusted analyses were extracted in preference over crude or age-
adjusted measures. In order of preference, risk ratios, HRs, incidence
ratios, and ORs were extracted.
1530 SHAMS-WHITE ET AL.
TABLE 1
Included bone outcomes of interest1
Outcome Sites or markers

BMC TB only
BMD TB, TH, FN, LS
Fractures All sites
Falls N/A
Bone quality For example, via Ad-SOS
Bone metabolism ALP, BAP, BSAP, CTX, NTX, C1NP or
biomarkers2 P1NP, DPD, hydroxyproline, OC, PYD
1
Ad-SOS, amplitude-dependent speed of sound; ALP, alkaline phosphatase; BAP, bone alkaline phosphatase; BMC,
bone mineral content; BMD, bone mineral density; BSAP, bone-specific alkaline phosphatase; CTX, collagen type 1 cross-
linked C-terminal telopeptide; C1NP, C-terminal type 1 procollagen; DPD, deoxypyridinoline; FN, femoral neck; LS,
lumbar spine; NTX, collagen type 1 cross-linked N-telopeptide; N/A, not applicable; OC, osteocalcin; P1NP, N-terminal
type 1 procollagen; PYD, pyridinoline; TB, total body; TH, total hip.
2
Ratios of the biomarkers (e.g., with creatinine) were included as outcomes of interest.

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Risk of bias in individual studies
We assessed the methodologic quality of each study on the basis
of predefined criteria. For intervention and prospective cohort
studies, we used a modified version of the Cochrane risk of bias
(ROB) tool (http://www.cochrane.org/handbook) and the New-
castle Ottawa Scale, respectively. Two independent investigators
conducted ROB assessments, with discrepancies discussed among
the research team and resolved via group consensus. Further details
can be found in Supplemental Methods 2.
Data synthesis
All of the included studies were summarized in narrative form
and in summary tables that tabulated key features of the study
populations, design, intervention, outcomes, and results. Summary
tables were organized by study type [i.e., randomized controlled trial
(RCT) compared with cohort study]. Results were quantitatively and
qualitatively summarized by study type and outcome of interest.
Qualitative synthesis
The strength of evidence (SOE) for major comparisons and
outcomes was assessed through a consensus process of the entire
research team, with the use of a modified version of a grading
system used by the American Diabetes Association and other
prominent groups (22, 23). Briefly, SOE levels could be categorized
as A (strong), B (moderate), C (limited), D (inadequate), E (expert
consensus or clinical experience), or NA (not applicable). Details of
the grading system can be found in Supplemental Table 2.
Meta-analysis (quantitative synthesis)
The methods outlined in the Cochrane Handbook for
conducting meta-analysis of RCTs were followed (24). Ob-
servational studies were not pooled due to insufficient data for
dose-response meta-analysis.
For BMD outcomes in RCTs, we used the reported or calculated
net percentage change between the higher- and lower-protein
groups as the effect size measure in the meta-analysis because
most RCTs reported within-group percentage changes in BMD
outcomes. The mean within-group percentage change, if not
reported, was calculated by using the postintervention mean minus
the baseline mean, then divided by the baseline mean and multiplied
by 100%. Its SD was estimated by using the SD of the mean change
divided by the baseline mean. The net percentage change was the
difference in the 2 within-group percentage changes by using the
lower-protein group as the reference group; thus, a positive net
percentage change indicates an effect (i.e., less bone loss) favoring
higher protein intake. The SD of the net percentage change was the
pooled SD of the 2 SDs of the mean change: SDpooled = Of[(n1 2 1) 3
SD1 + (n2 2 1) 3 SD2]/(n1 + n2 2 2)g, where n1 and n2 are the
sample sizes and SD1 and SD2 are the SDs of the mean within-
group percentage changes of the higher- and lower-protein groups,
respectively. For studies that did not report final sample sizes, the
baseline sample size was used in our calculations (25, 26).
For other outcomes [i.e., total body (TB) bone mineral content
(BMC), osteocalcin, and CTX], we used the reported or calculated
net change (difference of the 2 within-group changes from baseline)
between the higher- and lower-protein groups as the effect size
measure in the meta-analysis. If the SDs of the within-group changes
were not reported, the SD of the mean within-group change was
estimated by using the following formula: SDdiff = O(SDB2 + SDF2 –
2 3 Corr 3 SDB 3 SDF), where SDB is the SD at baseline and
SDF is the SD at the end of the study. We assumed a correlation
coefficient (Corr) value of 0.50 to impute the missing SD of the
mean within-group change. Sensitivity analyses that used Corr
values of 0.20 and 0.80 were conducted to evaluate the impacts of
the correlation assumptions on the meta-analysis results, and none
showed appreciable impacts (Supplemental Table 3).
Studies were excluded from meta-analyses if required information
for the aforementioned calculations was not reported for any given
outcome. The original authors were contacted to obtain missing
quantitative data that were needed for meta-analysis (26–31); 3
authors responded with the requested information (26, 28, 31).
In light of clinical heterogeneity (different doses and types of
protein interventions), we performed random-effects meta-
analyses for outcomes with $4 unique RCTs (32). We used
both the Q statistic (considered significant when P , 0.10) and
the I2 index to quantify the extent of statistical heterogeneity
(24). We defined low, moderate, and high heterogeneity as I2
values of 25%, 50%, and 75%, respectively. These cutoffs are
arbitrary and were used for descriptive purposes only (33).
All of the calculations and meta-analyses were conducted in
Stata SE 13 (StataCorp). The analytic data sets can be found in
the Supplemental Analytic Data Sets. Two-tailed P values
,0.05 were considered significant.
 SYSTEMATIC REVIEW OF PROTEIN AND BONE HEALTH
FIGURE 1 Literature search and study selection process. Cochrane Central Register of Controlled Trials, gateway.ovid.com; Global Health, www.ebscohost.com/
academic/global-health; Ovid Medline, gateway.ovid.com; Scopus, www.scopus.com; Web-of-Science, webofknowledge.com. RCT, randomized controlled trial.
RESULTS
Our search yielded 1999 citations: 1280 articles were identified
for dual abstract screening, of which 207 were identified for full-
text screening and 36 were included for data extraction (16 RCTs
and 20 prospective cohort studies). The details are summarized
in Figure 1. Results shown below are organized by question,
namely 1) the effect of higher compared with lower protein in-
take on bone health outcomes and 2) the synergistic effects of
Ca6D with protein on bone health outcomes. ROB assessments
can be found by study in Supplemental Tables 4 and 5.
Higher compared with lower protein intake
Lumbar spine BMD RCTs
Seven RCTs examined lumbar spine (LS) BMD (Supple-
mental Table 6) (26, 29, 34–38). Three studies prescribed
supplements (35–37). The doses of milk-based protein in each of
the higher-protein groups were 13.2 and 45 g/d for 7 d/wk and
30 g/d for 5 d/wk, and the dose in the lower-protein groups was
0 g/d. The other 4 studies prescribed dietary composition
changes (26, 29, 34, 38). The 4 higher-protein group in-
terventions consisted of .90 g protein/d, 25% and 30% of total
energy from protein/d, and 1.4 g protein $ kg21 $ d21 with 3
daily servings of dairy, whereas the lower-protein group’s in-
terventions consisted of ,80 g protein/d, 15% and 18% of total
energy from protein/d, and 0.8 g protein $ kg21 $ d21 with 2
daily servings of dairy, respectively. Studies were conducted
in different populations (Supplemental Table 6). Overall,
ROB was medium, primarily due to low compliance (,80%)
1531
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and incomplete outcome data (i.e., dropout rate .20%) (Sup-
plemental Figure 1, Supplemental Table 4).
Findings from the 7 RCTs were inconsistent: 3 RCTs (29, 36,
38) reported that the lower-protein groups lost significantly more
or gained significantly less LS BMD than the higher-protein
groups after 1 y, whereas 4 RCTs (26, 34, 35, 37) showed no
significant difference in LS BMD (Supplemental Table 7).
Our random-effects meta-analysis of 5 RCTs showed that
higher protein intake, on average, had a protective effect on LS
BMD compared with lower protein intake, increasing BMD by an
average of 0.52% with no statistical heterogeneity (pooled net
percentage change: 0.52%; 95% CI: 0.06%, 0.97%; I2: 0.0%)
(Figure 2). The meta-analysis was rerun without the study by
Kukuljan et al. (36), which had more than half the weight in the
meta-analysis, to see if the discrepancy was due to this one study
alone, and the pooled result was no longer significant (n = 4;
pooled net percentage change: 0.35%; 95% CI: 20.20, 0.89; I2:
0.0%; data not shown). Findings from the 2 studies that could
not be included in the meta-analysis were also inconsistent:
Thorpe et al. (29) found that those in the high-protein group
showed significantly higher LS BMD gain than did the low-
protein group after 1 y (data in figure only; P , 0.01),
whereas no differences between groups were found by Schürch
et al. (37) after 1 y (net difference; BMD T score: 0.010;
95% CI: 20.01, 0.03; P . 0.2) (Supplemental Table 7).
Cohort studies
Seven cohort studies examined LS BMD for 2.5–6 y (Sup-
plemental Table 8) (39–45). One cohort study comprised young,
1532 SHAMS-WHITE ET AL.
FIGURE 2 Effect of protein intake on lumbar spine bone mineral density. Weights are from random-effects analysis. Each gray box represents the individual
study’s effect estimate, and the horizontal line represents the 95% CI of the effect estimate. The diamond shape represents the meta-analysis pooled effect estimate
and its CI. A dotted vertical line displays the location of the meta-analysis pooled effect estimate. OWt, overweight; PM, postmenopausal women; Ref, reference.
nulliparous women (44), 1 included perimenopausal women (42),
1 comprised postmenopausal women (39), and the other 4 (40,
41, 43, 45) comprised both men and women [of which 2 (40, 41)
were in older adults only]. Protein intake was assessed by
using a 7-d diary in 1 study (44) and food-frequency questionnaires
(FFQs) in the remaining studies. Overall, ROB was low, except
most studies did not address if there was an adequate sample size or
power for any significant findings (Supplemental Table 5).
There was no significant association between protein intake
and LS BMD change in 6 of the 7 studies (Supplemental Table 7)
(39, 40, 42–45). Only 1 cohort study (41) conducted in older
adults found that those in the lowest protein category (7.3–
13.5% and 9.64–15.49% of total energy) showed the most LS
BMD loss, whereas those in the highest category (17.9–27.4%
of total energy) showed the least LS BMD loss.
Total hip BMD
RCTs
Eight RCTs examined total hip (TH) BMD (Supplemental Table
6) (26, 29, 31, 34–36, 38, 46). Four studies prescribed supplements
(31, 35, 36, 46). The doses of milk-based protein in the higher
protein groups ranged from 13.2 to 45 g/d, and in the lower protein
groups from 0 to 2.1 g/d. Four studies prescribed dietary compo-
sition changes (26, 29, 34, 38). In these 4 studies, the higher-protein
groups consumed .90 g protein/d, 25% and 30% of total energy
from protein/d, and 1.4 g protein $ kg21 $ d21 with 3 daily servings
of dairy, whereas the lower-protein groups consumed ,80 g pro-
tein/d, 15% and 18% of total energy from protein/d, and 0.8 g
protein $ kg21 $ d21 with 2 daily servings of dairy, respectively.
Studies were conducted in different populations (Supplemental Ta-
ble 6). Overall, ROB was medium, primarily due to low compliance
(,80%) and incomplete outcome data (i.e., dropout rate .20%)
(Supplemental Figure 1, Supplemental Table 4).
Six of the 8 RCTs (26, 31, 34–36, 46) found no significant
difference in the net changes in TH BMD over 1–2 y (Supplemental
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Table 7). Two RCTs (29, 38) that administered dietary compo-
sition changes found that the lower-protein groups lost signifi-
cantly more or gained significantly less TH BMD than the
higher-protein groups after 1 y (Supplemental Table 7).
Our random-effects meta-analysis of 7 RCTs showed similar
but smaller effects of higher compared with lower protein intakes
on TH BMD (pooled net percentage change: 0.30%; 95% CI:
20.02%, 0.62%; I2: 0.0%) (Figure 3). One study could not be
meta-analyzed: Thorpe et al. (29) reported that those in the
lower-protein group had significantly greater TH BMD loss than
those in the higher-protein group after 1 y (data in figure only;
P = 0.01) (Supplemental Table 7).
Cohort studies
Two cohort studies examined TH BMD for 4 and 6 y (Sup-
plemental Table 8) (39, 43). One cohort study comprised post-
menopausal women (39) and the second included adults aged
$55 y (43). Protein intake was assessed by using FFQs. ROB
was medium, mainly due to unclear power to detect a difference
in one study and the lack of completeness of the cohort follow-
up in both studies (e.g., .20% lost to follow-up or differential
loss of follow-up) (Supplemental Table 5). No significant asso-
ciation between protein intake and BMD change was found in
either study (Supplemental Table 9) (39, 43).
Femoral neck BMD
RCTs
Eight RCTs examined femoral neck (FN) BMD (25, 26, 31,
34–38) (Supplemental Table 6). Five studies (25, 31, 35–37)
prescribed supplements. The doses of milk-based protein in the
higher-protein groups ranged from 13.2 to 45 g/d, and in the
lower protein groups from 0 to 2.1 g/d. Three studies (26, 34,
38) prescribed dietary composition changes. In these 2 studies,
the higher-protein groups consumed .90 g protein/d, 25% or
 SYSTEMATIC REVIEW OF PROTEIN AND BONE HEALTH 1533

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FIGURE 3 Effect of protein intake on total hip bone mineral density. Weights are from random-effects analysis. Each gray box represents the individual
study’s effect estimate, and the horizontal line represents the 95% CI of the effect estimate. The diamond shape represents the meta-analysis pooled effect
estimate and its CI. A dotted vertical line displays the location of the meta-analysis pooled effect estimate. Fx, fracture; OWt, overweight; PM, post-
menopausal women; Ref, reference.

30% of total energy from protein, whereas the lower-protein
groups consumed ,80 g protein/d, 15% or 18% of total en-
ergy from protein, respectively. Studies were conducted in dif-
ferent populations (Supplemental Table 6). Overall, ROB was
medium, primarily due to low compliance (,80%) and in-
complete outcome data (i.e., dropout rate .20%) (Supplemental
Figure 1, Supplemental Table 4).
Seven of the 8 RCTs (26, 31, 34–38) found no significant dif-
ference in the net changes in FN BMD (Supplemental Table 7).
Only one study (25) in postmenopausal women with recent hip
fracture found that the lower-protein group lost significantly
more FN BMD in 1 y than the higher-protein group.
Our meta-analysis of 6 RCTs showed no difference in the
effects of higher compared with lower protein intake on FN
BMD, with no statistical heterogeneity (pooled mean percentage
change: 20.14%; 95% CI: 20.60%, 0.32%; I2: 0.0%) (Figure
4). Two studies (25, 37) could not be meta-analyzed: no sig-
nificant net difference in FN BMD was found between groups

FIGURE 4 Effect of protein intake on femoral neck bone mineral density. Weights are from random-effects analysis. Each gray box represents the
individual study’s effect estimate, and the horizontal line represents the 95% CI of the effect estimate. The diamond shape represents the meta-analysis pooled
effect estimate and its CI. A dotted vertical line displays the location of the meta-analysis pooled effect estimate. OWt, overweight; PM, postmenopausal
women; Ref, reference.
1534 SHAMS-WHITE ET AL.
after 1 y by Schürch et al. (37) (net difference; T score: 0.010;
95% CI: 0.000, 0.030; P = 0.111) or Tengstrand et al. (25) (net
difference; z score: 0.260; 95% CI: 20.046, 0.566; P . 0.05)
(Supplemental Table 7).
Cohort studies
Five cohort studies examined FN BMD for 2.5–4.6 y (Sup-
plemental Table 8) (40–43, 45). One cohort study (42) comprised
perimenopausal women and the other 4 comprised both men and
women [of which 2 (40, 41) were conducted in older adults only].
Protein intake was assessed by using FFQs. Overall, ROB was
low, except that no study addressed if there was adequate sample
size or power for any significant findings (Supplemental Table 5).
There was no significant association between protein intake
amounts and FN BMD change in 3 studies (Supplemental Table
9) (42, 43, 45). The 2 cohort studies (40, 41) conducted in older
adults found that those in the lowest protein category (7.3–13.5%
and 9.64–15.49% of total energy) showed the most FN BMD
loss, whereas those in the highest category (17.9–27.4% and
18.16–29.14% of total energy, respectively) showed the least FN
BMD loss (Supplemental Table 9).
TB BMD
RCTs
Five RCTs examined TB BMD (Supplemental Table 6) (25, 29,
38, 46, 47). Two studies (25, 46) prescribed supplements. The doses
of milk-based protein in the higher-protein groups were 20 and
40 g/d and those in the lower-protein groups were 0 g/d. The other 3
studies (29, 38, 47) prescribed dietary composition changes. The
higher-protein groups consumed 2.2 g protein $ kg lean body
mass21 $ d21, 30% of total energy from protein/d, and 1.4 g
protein $ kg21 $ d21 with 3 daily servings of dairy, whereas the
lower-protein groups consumed 1.1 g $ kg lean body mass21 $ d21,
18% of total energy from protein/d, and 0.8 g protein $ kg21 $ d21
with 2 daily servings of dairy, respectively. Studies were conducted
in different populations (Supplemental Table 6). Overall, ROB was
medium, primarily due to low compliance (,80%) and incomplete
outcome data (dropout rate .20%) (Supplemental Figure 1, Sup-
plemental Table 4).
Findings from the 5 RCTs were inconsistent: 2 RCTs (25, 29)
found that the lower-protein groups lost significantly more TB
BMD than did the higher-protein groups after 1 y; 3 RCTs (38,
46, 47) found no significant difference in the net changes in TB
BMD after 1 y (Supplemental Table 7). Data were not sufficient
to conduct a meta-analysis.
Cohort studies
Two cohort studies examined TB BMD for 3 and 6 y (Sup-
plemental Table 8) (39, 40). One cohort study (40) comprised
older adults and 1 study (39) comprised postmenopausal women,
respectively. Protein intake was assessed by using FFQs. ROB
was medium, primarily due to the lack of completeness of the
cohort follow-up in 1 study (i.e., .20% lost to follow-up)
(Supplemental Table 5). One study (39) found higher protein
intake to be associated with a significant increase in TB BMD
over time. The second study (40) found higher protein intake to
be associated with significantly less TB BMD loss over time com-
pared with lower protein intake, but only among those supplemented
with calcium and vitamin D; no such association was found
among those given a placebo (Supplemental Table 9).
TB BMC
RCTs
Three RCTs examined TB BMC (Supplemental Table 6) (29,
37, 38). One study (37) prescribed supplements: 20 g milk pro-
tein/d for 5 d/wk to the higher-protein group and 0 g protein to the
lower-protein group. The other 2 studies (29, 38) prescribed di-
etary composition changes. The higher-protein groups consumed
30% of total energy from protein/d and 1.4 g protein $ kg21 $ d21
with 3 daily servings of dairy, whereas the lower-protein
groups consumed 18% of total energy from protein/d and
0.8 g protein $ kg21 $ d21 with 2 daily servings of dairy, re-
spectively. All of the studies were conducted in different pop-
ulations (Supplemental Table 6). Overall, ROB was high,
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primarily due to low compliance in 2 of the studies (,80%) and
incomplete outcome data in all 3 studies (i.e., dropout rate
.20%) (Supplemental Table 4).
Findings from the 3 RCTs were inconsistent: 1 study (29) in
men found that the lower-protein group lost significantly more
TB BMC than did the higher-protein group after 1 y; 2 RCTs (37,
38) found no significant difference in the net changes in TB BMC
after 1 y (Supplemental Table 7). Data were not sufficient to
conduct a meta-analysis.
Cohort studies
One cohort study, which was composed of perimenopausal
women, examined TB BMC for 2.5 y (Supplemental Table 8)
(42). Protein intake from soy was assessed by using an FFQ.
Overall ROB for this study was medium, primarily due to .20%
of participants being lost to follow-up over the course of the
study (Supplemental Table 5). Higher protein intake was asso-
ciated with a significant increase in TB BMC over time com-
pared with lower protein intake (Supplemental Table 9) (42).
Falls
RCTs
No RCTs examining the risk of falls met the inclusion criteria.
Cohort studies
Two cohort studies in older adults examined the risk of falls
over a 1- to 2-y period (Supplemental Table 8) (48, 49). Protein
intake was assessed by using an FFQ. Overall ROB for these
studies was low, although it is important to note that it is unclear if
there was enough statistical power to detect an association with
continuous protein intake data in either study (Supplemental
Table 5). One study (49) found higher protein intake to be as-
sociated with a lower risk of falls compared with lower protein
intake, whereas the other study (48) found no significant asso-
ciation (Supplemental Table 10).
Spine fracture
RCTs
No RCTs examining spine fracture risk met the inclusion
criteria.
 SYSTEMATIC REVIEW OF PROTEIN AND BONE HEALTH
Cohort studies
One cohort study in postmenopausal women examined spine
fracture risk over a 6-y period (Supplemental Table 8) (39).
Protein intake was assessed by using an FFQ. The ROB for this
study was medium, primarily due to the completeness of the
cohort follow-up (i.e., .20% lost to follow-up) and selective
outcome reporting (Supplemental Table 5). There was no sig-
nificant association found between protein intake and spine
fracture risk (Supplemental Table 10) (39).
Hip fractures
RCTs
No RCTs examining hip fracture risk met the inclusion
criteria.
Cohort studies
Nine cohort studies examined hip fracture risk for 3–17 y
(Supplemental Table 8) (39, 50–57). The studies were conducted
in different populations: 3 studies (39, 50, 54) were conducted in
women [2 (39, 54) of which were conducted in postmenopausal
women], 2 (55, 57) were conducted in men, and 4 studies (51–
53, 56) were conducted in adults of both sexes. Protein intake
was assessed by using FFQs. Overall, ROB was low, although 3
studies adjusted for neither calcium nor vitamin D and sample
size adequacy was either unclear or not met in 5 studies (Sup-
plemental Table 5).
Six studies (39, 50, 52, 54–56) did not find a significant as-
sociation between protein intake and hip fracture risk (Supple-
mental Table 10). One study in men (57) found that increased
protein intake was associated with a decreased risk of hip
fracture. Another study (51) in adults stratified men and women
and only found a significant inverse association between higher
protein quartiles and the risk of hip fracture when compared
with the lowest quartile in women; there was no significant as-
sociation in men. Last, one study (53) found a significantly
lower risk of hip fracture only among participants in the third
quartile compared with those in the lowest quartile of protein
intake; no association was found in the second or highest
quartile (Supplemental Table 10).
Forearm fractures
RCTs
No RCTs examining forearm fracture risk met the inclusion
criteria.
Cohort studies
Two cohort studies examined forearm fracture risk for 6 and 12 y
(Supplemental Table 8) (39, 50). One cohort study comprised
postmenopausal women (39) and the other study (50) comprised
middle-aged women. Protein intake was assessed by using FFQs.
Overall, ROB was medium, because one study reported inadequate
sample size and power and self-reported fracture (50) and the other
study (39) had loss to follow-up (,80%) and incomplete outcome
reporting (Supplemental Table 5).
The 2 studies had opposite findings. In one study (50), which
compared the higher quintiles with the lowest quintile (,68 g/d),
the highest quintile (.95 g/d) showed a significant positive
1535
association between total protein and forearm fracture. Con-
versely, in the second study (39), when protein was examined in
quintiles by percentage of total energy, women who had 20%
higher protein intakes were 7% less likely to have a fracture
(Supplemental Table 10).
Overall fractures
RCTs
No RCTs examining overall fracture risk met the inclusion
criteria.
Cohort studies
Four cohort studies examined overall fracture risk for between
5 and 13 y (Supplemental Table 8) (39, 58–60). Three cohort
studies comprised postmenopausal women (39, 58, 59) and 1
comprised adults aged $55 y (60). Protein intake was assessed
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by using FFQs. Overall, ROB was low, although 3 studies
captured FFQ data only at baseline for $6 y of follow-up, and
2 studies used self-reported data for the outcome assessment
(Supplemental Table 5).
There was no significant association between protein intake
and overall fracture risk in 3 of the studies (Supplemental Table
10) (39, 58, 60). The fourth cohort study (59) found an inverse
association of overall fracture risk with higher soy protein intake
($4.98 g/d) with the use of the lowest quintile of soy protein
intake (,4.98 g/d) as the reference group.
Osteocalcin
RCTs
Ten RCTs examined osteocalcin concentrations (Supple-
mental Table 6) (25, 27, 28, 30, 34, 35, 37, 38, 61, 62). Eight
studies (25, 27, 28, 30, 35, 37, 61, 62) prescribed milk protein–
based supplements: the doses of milk-based protein in the
higher-protein groups in 7 studies ranged from 10 to 45 g/d and
1 study (62) supplemented 250-mg capsules of milk ribonuclease–
enriched lactoferrin/d, whereas doses in the lower-protein
groups ranged from 0 to 2.1 g milk-based protein/d. In the
higher-protein groups, 3 received 10–45 g milk-based protein/d,
3 received 40 mg milk-based protein/d, and 1 received 250 mg
milk lactoferrin capsules that were RNase-enriched. The lower-
protein groups received 0 g protein/d. The 2 remaining studies
(34, 38) prescribed dietary composition changes. The higher-
protein groups consumed .90 g protein or 30% of total en-
ergy from protein, whereas the lower-protein groups consumed
,80 g protein or 18% of total energy from protein, respectively.
Eight studies were conducted in different populations of women,
1 study was conducted in healthy adults, and 1 study was con-
ducted in adults with recent hip fracture (Supplemental Table 6).
Overall, ROB was low, although it is important to note that 4
studies had low compliance (,80%) and 4 studies had in-
complete outcome data (i.e., dropout rate .20%) (Supplemental
Figure 1, Supplemental Table 4).
Findings were inconsistent across studies: 6 of the 10 RCTs
(25, 27, 35, 37, 38, 61) found no significant difference in the net
changes in osteocalcin concentrations (Supplemental Table 11).
Two studies (30, 62) found that osteocalcin concentrations in-
creased significantly more in the higher-protein group than in
1536 SHAMS-WHITE ET AL.

the lower-protein groups, whereas another study found that os-
teocalcin concentrations decreased significantly more in the higher-
protein group than in the lower-protein group (34). Findings in
the remaining study (28) were unclear, because the higher-
protein group showed significantly different osteocalcin con-
centrations at both baseline and follow-up compared with the
lower-protein group, and final and change quantitative values
were not provided (i.e., data in figure only) (Supplemental
Table 11).
Our meta-analysis of 8 RCTs showed that higher protein in-
takes had no significant effects on osteocalcin compared with
lower protein intakes, with wide uncertainty (i.e., wide CIs) and
low statistical heterogeneity (pooled net change: 0.06 ng/mL;
95% CI: 20.49, 0.60 ng/mL; I2: 27.2%) (Figure 5). Two 6-mo
studies could not be included in the meta-analysis: Aoe et al.
(27) found no difference between protein groups (data in figure
only; P . 0.05), whereas Uenishi et al. (30) found the higher-
dietary composition changes. The higher-protein group consumed
.90 g protein/d, whereas the lower-protein group consumed
,80 g protein/d. Studies were conducted in different populations
(Supplemental Table 6). Overall, ROB was medium, primarily due
to low compliance (,80%) in 3 studies and incomplete outcome
data (dropout rate .20%) in 2 studies (Supplemental Figure 1,
Supplemental Table 4).
Three RCTs (25, 28, 46) found no significant difference in the
net changes in CTX concentrations (Supplemental Table 11). One
study (34) found that the lower-protein group showed signifi-
cantly greater increases in CTX concentrations compared with
the higher-protein group after 2 y, whereas another study (35)
found that the higher-protein group showed significantly higher
concentrations than the lower-protein group after 1.5 y (Sup-
plemental Table 11).
Our meta-analysis of 5 RCTs showed that higher protein in-
take, on average, increased CTX compared with lower protein

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protein group to have higher osteocalcin concentrations over
time compared with the lower-protein group (data in figure only;
P = 0.033) (Supplemental Table 11).
Cohort studies
The association between protein intake and osteocalcin con-
centrations was not examined for this outcome in any of the
included cohort studies.
CTX
RCTs
Five RCTs examined CTX concentrations (25, 28, 34, 35, 46)
(Supplemental Table 6). Four studies (25, 28, 35, 46) prescribed
supplements. The doses of milk-based protein in the higher-
protein groups ranged from 10 to 45 g/d, whereas the lower-
protein groups received 0 g protein/d. One study (34) prescribed
intake, although the pooled result was not significant, with wide
uncertainty (i.e., wide CIs) and substantial heterogeneity (pooled
net change: 47.72 ng/L; 95% CI: 227.34, 122.78 ng/L; I2:
61.3%) (Figure 6).
Cohort studies
The association between protein intake and CTX concentra-
tions was not examined for this outcome in any of the included
cohort studies.
Synergistic effects of protein and Ca6D
Most RCTs supplemented all of the participants with the same
or similar amounts of Ca6D, but none examined their syner-
gistic effects for our included outcomes of interest. The fol-
lowing results are thus from cohort studies only (Supplemental
Table 8). No studies examined the interaction between protein

FIGURE 5 Effect of protein intake on osteocalcin. Weights are from random-effects analysis. Each gray box represents the individual study’s effect
estimate, and the horizontal line represents the 95% CI of the effect estimate. The diamond shape represents the meta-analysis pooled effect estimate and its
CI. A dotted vertical line displays the location of the meta-analysis pooled effect estimate. Fx, fracture; OWt, overweight; PM, postmenopausal women; Ref,
reference.
 SYSTEMATIC REVIEW OF PROTEIN AND BONE HEALTH
FIGURE 6 Effect of protein intake on C-terminal telopeptide. Weights are from random-effects analysis. Each gray box represents the individual study’s
effect estimate, and the horizontal line represents the 95% CI of the effect estimate. The diamond shape represents the meta-analysis pooled effect estimate and
its CI. A dotted vertical line displays the location of the meta-analysis pooled effect estimate. Fx, fracture; PM, postmenopausal women; Ref, reference.
and Ca6D on TB BMC, falls, osteocalcin, or CTX and thus we
conclude there is D level of evidence for these outcomes.
BMD
Four (39, 40, 43, 45), 2 (39, 43), and 3 (40, 43, 45) cohort
studies examined the interaction between protein and Ca6D on
LS BMD, TH BMD, and FN BMD outcomes, respectively
(Supplemental Table 8). Overall, ROB was medium for all 3
BMD outcomes, primarily due to loss to follow-up (.20%) and
not addressing if there was adequate sample size or power to
detect significant findings in 3 studies (Supplemental Table 5).
All except for one of the studies (40) did not find a significant
interaction (Supplemental Table 9). This study (40) was a cohort
study that used data from a 3-y, randomized, placebo-controlled
trial with or without Ca6D supplementation and found that
higher dietary protein intake was associated with less TB BMD
loss than was lower protein intake only in the group supple-
mented with Ca6D (Supplemental Table 9).
Spine fractures
One cohort study examined the interaction between protein
and calcium (Supplemental Table 8) (39), but its findings were
not significant (Supplemental Table 10). Overall ROB for this
study was medium, primarily due to loss to follow-up (.20%)
and incomplete reporting for this outcome (Supplemental
Table 5).
Hip fractures
Four cohort studies also examined an interaction between
protein and calcium intake (39, 52, 55, 56) (Supplemental Table
8). Overall, ROB was medium, primarily due to 2 studies that
lost .20% of their subjects during follow-up, 4 studies only
ascertaining nutrient exposure at baseline for $6 y of follow-
up, and 3 studies either not addressing or not having an adequate
1537
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sample size or power to detect significant findings (Supplemental
Table 5). Of these 4 studies, 2 (39, 55) did not find a significant
interaction. One study (56) did find a significant interaction be-
tween protein and calcium, but once stratified by calcium, find-
ings were not significant for either of the calcium categories. The
fourth study (52) reported on men and women separately and
found an increased risk of hip fracture only among women with
both the highest quartile of protein intake and the lowest quartile
of calcium intake (Supplemental Table 10).
Forearm fractures
Two cohort studies examined an interaction between protein
and calcium intake (39, 50) (Supplemental Table 8), but neither
of these studies found a significant interaction (Supplemental
Table 10). Overall, ROB was medium, because one study (50)
reported an inadequate sample size and power and self-reported
fracture and the other study (39) had loss to follow-up (.20%)
and incomplete reporting for this outcome (Supplemental
Table 5).
Overall fractures
Two cohort studies examined an interaction between protein
and dietary calcium intake, with inconsistent findings (Supple-
mental Table 8) (39, 58). Overall, ROB was low, although both
studies only captured FFQ data at baseline for $6 y, and 1 study
used self-reported data for the outcome assessment (Supple-
mental Table 5). One study (39) did not find a significant in-
teraction between protein and calcium. The second study (58)
found that, when the lowest protein quartile plus the lowest
calcium quartile was used as the reference group, a higher
protein intake (g/1000 kcal) was positively associated with
overall fracture risk in the lowest calcium quartile. It was not
significantly associated with overall fracture risk in the higher
calcium quartiles (Supplemental Table 10).
1538 SHAMS-WHITE ET AL.
TABLE 2
SOE grading: higher compared with lower protein intake by outcome1
Studies, n (ref)

Outcome RCTs Cohort studies SOE grade Explanation

BMD
LS 7 (26, 29, 34–38) 7 (39–45) B We conclude a B level of evidence that a higher protein
intake may cause less LS BMD loss than lower protein
intake in older adults. Three RCTs (2 in older men and 1 in
postmenopausal women) found this effect, whereas 4 (2 in
healthy adults, 1 in postmenopausal women, and 1 in older
adults with current hip fracture) found that protein intake
had no significant effect on LS BMD. Studies in various
populations are currently limited, varying doses and
dietary compositions were used or prescribed,
respectively, and there was medium ROB among the
RCTs. Cohort studies overall did not support this
association between higher protein intake and LS BMD

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loss, although there may be ROB because of loss to
follow-up or because it is unclear if they had adequate
sample size and power to detect an association.
TH 8 (26, 29, 31, 34–36, 38, 46) 2 (39, 43) B We conclude a B level of evidence that protein intake amount
does not affect TH BMD loss. Most of the RCTs did not
find protein intake to significantly affect TH BMD in
various populations, and only 2 studies found a difference.
Studies in each population are also limited, varying doses
were used, and there was medium ROB. Two cohort
studies also found no association between protein intake
and TH BMD loss, although it is important to note that the
number of cohort studies included were limited with
medium ROB.
FN 8 (25, 26, 31, 34–38) 5 (40–43, 45) B We conclude a B level of evidence that protein intake amount
does not affect FN BMD loss. Although RCTs overall did
not find protein intake to significantly affect FN BMD in
various populations, studies in each population are
limited, varying doses were used, and there was medium
ROB. Some cohort studies found no association between
higher protein intake and BMD loss, although 2 in older
adults reported high protein intake to be associated with
less BMD loss.
TB 5 (25, 29, 38, 46, 47) 2 (39, 40) B We conclude a B level of evidence that higher protein intake
may cause less TB BMD loss than lower protein intake.
Two RCTs (1 in postmenopausal women with FN
fractures and 1 in men) found this effect, whereas 3 (1 in
postmenopausal women, 1 in adults with recent hip
fracture, and 1 in adults) found that protein intake had no
significant effect on TB BMD. Multiple studies in various
populations are currently limited, varying doses and
dietary compositions were used or prescribed,
respectively, and there was medium ROB among the
RCTs. Cohort studies overall supported this association
between higher protein intake and less TB BMD loss.
TB BMC 3 (29, 37, 38) 1 (42) C No firm conclusions can currently be drawn. One RCT and 1
cohort study support the hypothesis that higher protein
intake causes less TB BMC loss than lower protein intake,
whereas 2 RCTs support the hypothesis that protein intake
has no significant effect on TB BMC. Furthermore,
varying protein doses were used in each RCT intervention
group, studies in various populations are limited, and there
was medium ROB among both the RCTs and cohort study.
(Continued)
 SYSTEMATIC REVIEW OF PROTEIN AND BONE HEALTH 1539
TABLE 2 (Continued )

Studies, n (ref)

Outcome RCTs Cohort studies SOE grade Explanation

Falls 0 2 (48, 49) D There are insufficient data to support a hypothesis. Only 2
cohort studies in middle-aged and older adults examined
this association: one supports the hypothesis that higher
protein intake is associated with lower risk of falls,
whereas the other found no significant association.
Fractures
Spine 0 1 (39) D There are insufficient data to support a hypothesis. Only one
cohort study in postmenopausal women examined this
association and found no significant association.
Hip 0 9 (39, 50–57) B We conclude a B level of evidence that protein intake amount
is not associated with hip fracture risk due to the multiple
cohort studies with a null finding and an overall low risk of
bias.

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Forearm 0 2 (39, 50) D We conclude a D level of evidence that higher protein intake
is associated with forearm fracture compared with lower
protein intake. Although 2 cohort studies found
a significant association between higher protein intake and
forearm fracture, the results were inconsistent. In addition,
ROB could have affected findings. One study reported low
power due to low rates of fractures in their population
(50), whereas the second study (39) may have had bias due
to loss to follow-up (,80%).
Overall 0 4 (39, 58–60) D We conclude a D level of evidence that protein intake
amount is associated with overall fracture risk due to
a limited number of studies and conflicting results.
Although 1 cohort study found an inverse association
between higher soy protein intake and fracture risk, 3
studies found no association.
Osteocalcin 10 (25, 27, 28, 30, 34, 35, 37, 38, 61, 62) 0 B We conclude a B level of evidence that protein intake amount
does not affect osteocalcin concentrations. Six RCTs did
not find protein intake to significantly affect osteocalcin
concentrations in various female populations, 1 study was
unclear, and 3 studies had different findings on the
directionality of the effect of higher protein intake on
osteocalcin concentrations. ROB was low overall.
CTX 5 (25, 28, 34, 35, 46) 0 B We conclude a B level of evidence that protein intake amount
does not affect CTX concentrations. Three RCTs did not
find protein intake to significantly affect CTX
concentrations in various populations and 2 studies had
opposing findings. Studies in each population are limited,
varying doses were used, and there was low compliance in
2 of the 4 studies.
1
BMC, bone mineral content; BMD, bone mineral density; CTX, collagen type 1 cross-linked C-terminal telopeptide; FN, femoral neck; LS, lumbar
spine; RCT, randomized controlled trial; ref, reference; ROB, risk of bias; SOE, strength of evidence; TB, total body; TH, total hip.

SOE grading With regard to biochemical markers of bone turnover, an evidence

An evidence level of B was assigned for the association between
higher compared with lower protein intakes and BMD of the LS,
TH, FN, and TB, with no effect for the TH and FN; possibly less
BMD loss for the LS; and inconclusive effects for the TB. An
evidence level of C was assigned for the association of higher
compared with lower protein intakes and total body BMC, with no
firm conclusions possible. With regard to fracture outcomes, an
evidence level of B was assigned for no relation between higher
compared with lower protein intakes and hip fractures, but a level
of D was assigned for fractures of other sites (i.e., spine, forearm,
and overall fracture risk) due to insufficient or inconsistent data. An
evidence level of D was assigned for falls due to insufficient data.
level of B was assigned for no relation between higher compared
with lower protein intakes and osteocalcin or CTX (Table 2).
An evidence level of C was assigned for no synergistic effect
between protein and Ca6D on LS BMD, TH BMD, and forearm
fractures, as well for the inconclusive effect on FN BMD, TB
BMD, and overall fractures. An evidence level of D was as-
signed for all remaining outcomes due to insufficient data to
support a hypothesis (Table 3).
DISCUSSION
The relation between dietary protein intake and bone health
has been a topic of great debate over the past several decades.
1540 SHAMS-WHITE ET AL.
TABLE 3
SOE grading: synergistic effect of protein with calcium with or without vitamin D by outcome1
Studies, n (ref)

Outcome RCTs Cohort studies SOE grade Explanation

BMD
LS 0 4 (39, 40, 43, 45) C We conclude a C level of evidence that there is no synergistic
effect of protein with calcium with or without vitamin D
on FN BMD loss. Most of the cohort studies included
addressed this association with no significant findings,
although there may be ROB because of loss to follow-up
or because it is unclear if they had adequate sample size
and power to detect an association.
TH 0 2 (39, 43) C We conclude a C level of evidence that protein intake does
not have a synergistic effect with calcium on TH BMD
loss. Although 2 cohort studies did not find this interaction
to be significant, the power to detect an association was
unclear in 1 study and we have concerns with .20% of

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participants being lost to follow-up in both studies.
FN 0 3 (40, 43, 45) C No firm conclusions can currently be drawn. Only a few
cohort studies addressed this association with inconsistent
findings, and it is unclear if they had adequate sample size
and power to detect an association.
TB 0 2 C No firm conclusions can currently be drawn. Only 2 cohort
studies addressed this association with inconsistent
findings.
TB BMC 0 0 D There are insufficient data to support a hypothesis: no study
examined this association.
Falls 0 0 D There are insufficient data to support a hypothesis: no study
examined this association.
Fractures
Spine 0 1 (39) D There are insufficient data to support a hypothesis: only one
cohort study examined this association.
Hip 0 4 (39, 52, 55, 56) D There are insufficient data to support a hypothesis. There are
inconsistent results among the limited number of studies
that addressed this possible interaction between protein
and calcium.
Forearm 0 2 (39, 50) C We conclude a C level of evidence that there is no synergistic
effect of protein with calcium with or without vitamin D
on forearm fractures. Two cohort studies addressed this
association with no significant findings. However, there
may be ROB because it is unclear if they had adequate
sample size and power to detect an effect in one study, or
because of loss to follow-up in the other study.
Overall 0 2 (39, 58) C No firm conclusions can currently be drawn. Only 2 cohort
studies addressed this association with inconsistent
findings, and it is unclear if the existing studies had
adequate sample size and power to detect synergistic
effects, or adequate follow-up time to accurately capture
fracture risk.
Osteocalcin 0 0 D There are insufficient data to support a hypothesis: no study
examined this association.
CTX 0 0 D There are insufficient data to support a hypothesis: no study
examined this association.
1
BMC, bone mineral content; BMD, bone mineral density; CTX, collagen type 1 cross-linked C-terminal telopeptide;
FN, femoral neck; LS, lumbar spine; RCT, randomized controlled trial; ref, reference; SOE, strength of evidence; TB, total
body; TH, total hip.

There is an apparent dietary requirement for protein to build and
maintain bone; however, reports of detrimental effects of protein
made this more in-depth analysis necessary to better inform the
clinical community and stimulate additional research. Our
analysis was limited to direct measures in terms of fracture
outcomes, BMD, BMC, and select bone turnover markers. A
reductionist approach is often used (particularly in meta-analyses)
to assess the impact of a particular food or nutrient on a health
endpoint or disease outcome. Although this approach is useful in
guiding us in establishing reference intakes, it is often confounded
by the many synergies that exist with other essential nutrients that
promote healthy cell function.
 SYSTEMATIC REVIEW OF PROTEIN AND BONE HEALTH
Although the scientific literature is somewhat limited with
regard to the beneficial effects of protein intake, our analysis does
not indicate the presence of any adverse relations. Only a small
number of studies indicate an adverse association of protein
intakes and bone health. Positive trends for the relation of high
compared with low protein intakes and BMD and/or BMC were
identified for nearly all bone sites, although many findings were
inconsistent across studies. An evidence level of B was assigned
for the association between higher compared with lower protein
intakes and BMD of the LS, TH, FN, and TB, with no effect for
TH and FN and inconclusive effects for LS and TB. An evidence
level of C was assigned for the association of higher compared
with lower protein intakes and total body BMC, with no firm
conclusions possible. An evidence level of D was assigned for
falls due to insufficient data. With regard to biochemical markers
of bone turnover, an evidence level of B was assigned for no
relation between higher compared with lower protein intakes and
osteocalcin or CTX (Table 2).
With regard to fracture outcomes, an evidence level of B was
assigned for no relation between higher compared with lower
protein intakes and hip fractures; however, a level of D was
assigned for fractures of other sites (i.e. spine, forearm, and
overall fracture risk) due to insufficient or inconsistent data. Thus,
our study of limited data found no significant beneficial or
detrimental effects on high compared with low protein intakes
and fracture outcomes. In the absence of long-term intervention
studies, however (most study durations were #2 y), it is difficult
to assess at this time whether high compared with low protein
intakes affect fracture risk. Because fractures indeed represent
the most important outcome from a clinical standpoint, further
study is warranted.
Furthermore, significant interactions between dietary protein
with Ca6D intake were not shown in the included prospective
cohort studies (Table 3). However, it is important to reiterate the
limited number of cohort studies included and the level C
(i.e., “limited”) and level D (i.e., “inadequate”) SOE gradings
across all the outcomes. Because Ca6D supplementation has
recently been shown to significantly decrease the risk of both hip
and total fractures (63), further studies are merited.
Our systematic review and meta-analysis has several caveats in
addition to the inherent limitations related to the ROB from the
included studies. First, RCTs reported diverse BMD outcome
metrics, which make meta-analysis very challenging. Some
studies did not report sufficient data for meta-analysis, and
several assumptions were made to impute missing SDs of the
effect sizes, further limiting our confidence in the meta-analysis
results. Second, there is unaccountable clinical heterogeneity
(e.g., different protein intervention doses and comparators) in our
meta-analyses, and the small number of RCTs for each outcome
did not allow us to perform subgroup meta-analyses or meta-
regression to quantitatively examine the influences of clinical
heterogeneity on our pooled results. We were also unable to
identify apparent reasons to explain the inconsistent findings
across RCTs through qualitative evaluations, due to the diversity
of population characteristics. However, we considered all of these
limitations, including clinical heterogeneity, in our qualitative
SOE synthesis. Third, many RCTs had low adherence, whereas
some cohort studies experienced loss to follow-up and many did
not report if they had adequate sample sizes and power. The
presence of these biases across studies may have influenced the
1541
results. Finally, intake estimates from the observational studies
relied on 24-h dietary recalls and/or FFQs, which have known
limitations due to measurement errors. The measurement errors
may have had an impact on the results, but the directions of these
impacts are unpredictable.
Conclusions are further limited by the clear heterogeneity
present across studies. Studies included in this systematic review
used a variety of designs, doses, durations, and outcomes. Many
studies also may not have been long enough to see clear effects on
BMD, BMC, and fractures. Thus, future large RCTs that span
many years in duration are needed to accurately assess the effect
of protein intakes on fracture risk, along with surrogate endpoints
(e.g., BMD) on bone health. Additional, well-designed cohort
studies may also facilitate better insight, because large RCTs are
expensive and often not possible.
In conclusion, although positive trends for the relation of high
compared with low protein intakes and BMD and/or BMC were
Downloaded from ajcn.nutrition.org at BOSTON UNIVERSITY on January 2, 2018
identified for nearly all bone sites, only the LS had a moderate
SOE, showing that a higher protein intake may cause less BMD
loss than a lower protein intake in older adults. However, studies
were highly heterogeneous and confounding by variables that
were not accounted for could not be excluded when hypothe-
sizing why many outcomes were not significant. We found no
significant relation between dietary protein and fracture risk in
this systematic review. Larger, long-term RCTs are greatly
needed to clarify the role of dietary protein and fracture risk and
BMD changes. Overall, the body of evidence shows that the
effect of dietary protein on the skeleton appears to be favorable
to a small extent and is not detrimental. However, the existing
data are too heterogeneous and the SOE is not strong enough to
warrant a clinical guideline to recommend an increase in protein
intake as a standard care protocol.
We thank Amy Lapidow and Amy LaVertu for aiding MMS-W in the de-
velopment of all search strategies. We also thank Lars Holm, Kun Zhu, and
Russell de Souza for providing original data from their research studies upon
request for inclusion in the meta-analyses.
The authors’ responsibilities were as follows—MMS-W, MC, KLI, MSL,
SAS, TCW, and CMW: designed the research, developed the overall research
plan, and interpreted the results; MC and TCW: conceived of the project
conception and obtained funding; MMS-W: was responsible for study over-
sight; MMS-W, MD, ZF, MC, MCK, and JS: conducted the research; MMS-W,
MC, KLI, MCK, MSL, SAS, JS, and CMW: conducted qualitative
synthesis; MMS-W, MC, and ZF: conducted quantitative analyses; MMS-W,
KLI, and TCW: wrote the manuscript; MMS-W, MC, KLI, MCK, MSL,
SAS, TCW, and CMW: had primary responsibility for the final content;
and all authors: read and approved the final manuscript. TCW is a consultant
for the National Osteoporosis Foundation. None of the other authors report a
conflict of interest. MC, MD, KLI, MSL, SAS, and CMW contributed their
efforts without receiving funding or salary support. The Egg Nutrition Cen-
ter and Dairy Management Inc. had no role in the design, analysis, interpre-
tation, or presentation of the data or the results.
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