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Food & Nutrition Research 2023. © 2023 Christel Lamberg-Allardt et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose ,
even commercially, provided the original work is properly cited and states its license. Citation: Food & Nutrition Research 2023, 67: 9003 - http://dx.doi.org/10.29219/fnr.v67.9003
research
food
& nutrition
REVIEW ARTICLE
Animal versus plant-based protein and risk of cardiovascular
disease and type 2 diabetes: a systematic review of randomized
controlled trials and prospective cohort studies
Christel Lamberg-Allardt1, Linnea Bärebring2, Erik Kristoffer Arnesen3, Bright I.
Nwaru4,Birna Thorisdottir5, Alfons Ramel6, Fredrik Söderlund7, Jutta Dierkes8,
AgnetaÅkesson7
1Department of Food and Nutrition, University of Helsinki, Finland; 2Department of Internal Medicine and
Clinical Nutrition, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Sweden; 3Department
of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Norway; 4Krefting Research Centre,
Institute of Medicine, University of Gothenburg, Sweden; 5Health Science Institute, University of Iceland,
Iceland; 6Faculty of Food Science and Nutrition, University of Iceland, Iceland; 7Unit of Cardiovascular and
Nutritional Epidemiology, Institute of Environmental Medicine, the Karolinska Institute, Sweden; 8Centre for
Nutrition, Department of Clinical Medicine, University of Bergen, Norway and Department of Laboratory
Medicine and Pathology, Haukeland University Hospital, Norway
Abstract
Objectives: To systematically review the evidence on the effect of replacing the intake of animal protein with
plant protein on cardiovascular disease (CVD) and type 2 diabetes (T2D) and their intermediate risk factors.
Methods: We searched MEDLINE, Embase, Cochrane Central Register of Controlled Trials, and Scopus
up to 12th May 2022 for randomized controlled trials (RCTs) or prospective cohort studies that investigated
replacement of animal protein with plant protein from foods. Outcomes were CVDs, T2D, and in RCTs
also the effects on blood lipids, glycemic markers, and blood pressure. Risk of bias was evaluated with the
Cochrane’s RoB2, ROBINS-I, and USDA’s RoB-NObS tools. Random-effects meta-analyses assessed the
effects of plant vs. animal proteins on blood lipids in RCTs. The evidence was appraised according to the
World Cancer Research Fund’s criteria.
Results: After screening 15,090 titles/abstracts, full text of 124 papers was scrutinized in detail, from which
13 RCTs and seven cohort studies were included. Eight of the RCTs had either some concern or high risk
of bias, while the corresponding evaluation of cohort studies resulted in moderate risk of bias for all seven.
Meta-analyses of RCTs suggested a protective effect on total cholesterol (mean difference -0.11 mmol/L; 95%
CI -0.22, -0.01) and low-density lipoprotein cholesterol (-0.14 mmol/L; 95% CI -0.25, -0.02) by replacing ani-
mal protein with plant protein. The substitution of animal protein with plant protein (percentage of energy
intake) in cohort studies was associated with lower CVD mortality (n = 4) and lower T2D incidence (n = 2).
The evidence was considered limited-suggestive for both outcomes.
Conclusion: Evidence that the substitution of animal protein with plant protein reduces risk of both CVD
mortality and T2D incidence is limited-suggestive. Replacing animal protein with plant protein for aspects of
sustainability may also be a public health strategy to lower the risk of CVD mortality and T2D.
Popular scientic summary
• This systematic review on animal vs. plant protein and cardiovascular disease (CVD), type-2 dia-
betes (T2D), and cardiometabolic risk factors comprised cohort studies with substitution models
and interventions with replacement.
• The evidence linking substitution of animal with plant protein to lower CVD mortality and T2D
incidence was deemed limited-suggestive.
• Replacement of animal protein with plant protein for sustainability may also be considered as a
public health strategy to lower the risk of CVD and T2D.
Citation: Food & Nutrition Research 2023, 67: 9003 - http://dx.doi.org/10.29219/fnr.v67.9003
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Christel Lamberg-Allardt et al.
The role of protein intake and its effect on health out-
comes has been a long-standing research topic of inter-
est and has been a high priority in nutrition research
and disease prevention. In addition, efforts to combat climate
change have identied protein intake as an important target,
especially reducing protein of animal origin, since the pro-
duction of animal protein generally is resource-intensive and
environmentally impactful compared to plant protein sources
(1). Compared to plant protein, animal protein sources are
generally associated with larger carbon footprints, more land
use, and larger blue water footprints (2).
Cardiovascular disease (CVD) and type 2 diabetes
(T2D) are the major causes of morbidity and mortality
worldwide and are associated with high societal costs (3).
A recent systematic review (SR) and meta-analysis of
observational studies indicated that habitual high intake
of total and animal protein is associated with an increased
risk of T2D (4). In contrast, Mousavi et al. (5) showed
no association between dietary protein intake from dif-
ferent sources and risk of CVD in an SR of prospective
studies. Likewise, in another recent SR, dietary protein
intake from different sources showed no association with
risk of coronary heart disease (CHD), but in subgroup
analysis, there was a lower risk of CVD mortality with
an increasing plant protein intake (6). The latter observa-
tion was further supported in an SR by Qi et al. (7) who
demonstrated that higher plant protein intake was associ-
ated with a reduced risk of all-cause and CVD mortality.
Equally, Chen et al. (8) presented evidence from prospec-
tive cohort studies that suggested that total protein intake
was associated with an increased risk of all-cause mortal-
ity, mainly driven by an increased risk of CVD mortality
by intake of animal protein. However, this SR showed
that plant protein intake was inversely associated with
all-cause and CVD mortality. The SR performed for the
2012 Nordic Nutrition Recommendations (NNR) on pro-
tein intake and several outcomes, including CVD, body
weight, cancer, T2D, fractures, renal outcomes, physical
training, muscular strength, and mortality concluded that
many of the included studies found benecial associations
with plant protein intake (9).
In revising the NNR for the 2022 edition, the intake of
animal protein vs. plant protein in adults was a prioritized
subject by the NNR Committee for an SR. Criteria for
shortlisting topics were published in 2021 (10). Briey, it
was deemed justied to perform a new SR if there were
important new scientic data since NNR 2012 and no
recent, relevant, and qualied SR available on the topic
(11). A scoping review identied new data since 2011 that
may be relevant. The aim of this SR was to examine the
evidence for whether replacing animal protein with plant
protein reduces the risk of CVD and T2D.
Methods
The methodology for the present SR followed the
guidelines developed for the NNR 2022 (12, 13) and
the Preferred Reporting Items for SRs and Meta-
Analyses (14, 15). A protocol was pre-registered online
on PROSPERO (https://www.crd.york.ac.uk/prospero,
CRD42021240630). A focused research question was
developed by the NNR 2022 Committee, dening the
population/participants, intervention/exposure, con-
trol, outcome, timeframe, study design, and setting (PI/
ECOTSS), in an iterative process with the SR authors.
The funding source for NNR 2022 was the Nordic
Council of Ministers and governmental food and health
authorities of Norway, Finland, Sweden, Denmark, and
Iceland (10).
Eligibility criteria
The inclusion and exclusion criteria are outlined in the PI/
ECOTSS statement (Table 1). Briey, prospective cohort
studies and non-randomized and randomized controlled
trials (RCTs) conducted in healthy adult populations (>18
years) were eligible for inclusion. Studies including sub-
jects with mild hypercholesterolemia (as reported by the
authors), who were not treated with cholesterol-lowering
medication, were included in the analyses of RCTs. We
excluded prospective cohort studies that did not report on
substitution of animal protein with plant protein in rela-
tion to the outcomes, and those that were from settings
otherwise not relevant for the Nordic/Baltic population.
In this case, studies that evaluated a parallel compari-
son between the intake of animal and plant protein were
excluded as no substitution was performed in such stud-
ies. For RCTs using soy protein as plant protein source, we
included only RCTs intervening soy with zero or low iso-
avone content and excluded those with moderate or high
isoavone content. For interventions using soy protein
with different levels of isoavones, only the group with
To access the supplementary material, please visit the article landing page
Keywords: dietary protein; plant protein; cardiovascular disease mortality; incidence of type 1 diabetes; blood lipids
Received: 7 September 2022; Revised: 3 February 2023; Accepted: 8 February 2023; Published: 28 March 2023
Citation: Food & Nutrition Research 2023, 67: 9003 - http://dx.doi.org/10.29219/fnr.v67.9003 3
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Animal versus plant-based protein and risk of CVD and T2D
the lowest isoavone content was included to discount
effects of isoavones and focus on those of the protein
(16). Outcomes included CVD (mortality and incidence),
T2D, and related cardiometabolic risk factors.
Information sources and search strategy
A comprehensive literature search of MEDLINE (Ovid),
Embase (Ovid), Cochrane Central Register of Controlled
Trials, and Scopus was performed by a research librarian
from the Karolinska Institutet University Library up to the
search date, initially on 26th–28th March 2021, updated
on 12 May 2022. The search strategy (Supplementary le
1) was developed in collaboration with the authors, led by
CL-A and LB, and was peer-reviewed by research librar-
ians at the University of Oslo Library of Medicine and
Science, Norway. There were no date or language limita-
tions in the search strategy. Grey literature searches were
not performed.
Selection and data collection process
Two investigators (JB and BN) independently reviewed
titles, abstracts, and full-text articles for inclusions
according to the PI/ECOTSS statement (Table 1), rst in a
pilot test of 10% of the papers, using the web tool Rayyan
(https://rayyan.qcri.org) in blinded mode. Potentially eli-
gible papers were retrieved and read in full text by the
same two reviewers. Disagreements about inclusion were
resolved by a third reviewer (AÅ).
Another four authors (JD, EA, AR, and FS), in pairs,
independently extracted data from the included studies
into pre-specied Excel forms. Disagreements were solved
by discussion. Among the variables extracted were study
design, information on recruitment, dietary intake, inter-
ventions and controls, assessment of outcomes, follow-up,
drop-out, confounders, etc.
Study risk of bias assessment
Risk of bias in each included study was assessed by two
authors (CLA and BT), working independently. The
assessment tools used were Cochrane’s Risk of Bias 2.0
(17) and Risk of Bias In Non-randomized Studies of
Interventions (18, 19) for intervention studies, while ‘Risk
of Bias for Nutrition Observational Studies’ (RoB-NObS)
(20) was used for prospective observational studies. The
risk of bias in each individual study was classied as ‘low’,
‘some concerns’, or ‘high’. Risk of bias was visualized by
using the web app Risk-of-bias VISualization (robvis)
(21).
Synthesis methods
We performed a qualitative synthesis of the included
studies by describing the main characteristics. Following
the recommendations of the Healthcare Research and
Quality (AHRQ), the Cochrane Handbook, and the
NNR 2022 Handbook, a meta-analysis was performed if
>3 independent RCTs or >5 cohort studies were available
(12, 22–24).
Consequently, quantitative syntheses were per-
formed of RCTs reporting effects on total cholesterol,
LDL-cholesterol, HDL-cholesterol, and triglycerides.
Measures expressed in mg/dl were converted to mmol/l
by dividing mg/dl by 38.67 for cholesterol and 88.57
mg/dl for triglycerides. We used the random-effects
meta-analyses with variance (τ2) estimated by the
restricted maximum likelihood method. For most
parallel-group and crossover trials, we used pooled
differences in means and standard deviations (SD) of
follow-up values, while if post-intervention outcomes
were not reported, we included change from baseline
scores. The SDs were imputed from standard errors
if not reported. Homogeneity was assessed by the
Table 1. Eligibility criteria for population/participants, intervention/exposure, control, outcome, timeframe, study design, and settings
Plant vs animal protein
Population Intervention or
exposure
Comparators Outcomes Timing Setting Study design
Adults, 18 years
orolder
Plant protein
intake
Animal protein
intake
Atherosclerotic CVD including:
Major incident fatal and non-fatal
CVD(combined or separate:
myocardialinfarction, stroke,
coronaryheart disease, and
coronary artery bypass graft)
CVD mortality
Incident T2D
Changes in insulin resistance, insu-
lin sensitivity, HBA1c, fasting
glucose, and insulin
Changes in blood pressure and
bloodlipids
Intervention
trials must
have ≥4 weeks
of follow-up
and cohorts
>12 months
of follow-up
Relevant for
the general
population in
theNordic
and Baltic
countries
Randomized or
non-random-
ized interven-
tion trials
For obser-
vational
epidemiological
studies, we
will consider
prospec-
tive cohort
studies, nested
case–control
studies, and
case–cohort
studies
CVD, cardiovascular disease; T2D, type 2 diabetes; HbA1c, hemoglobin A1c.
Citation: Food & Nutrition Research 2023, 67: 9003 - http://dx.doi.org/10.29219/fnr.v67.9003
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Christel Lamberg-Allardt et al.
Cochran Q test, and we used the I2 statistic to quan-
tify variability explained by between-study heterogene-
ity. I2 of ≥50% was considered ‘substantial’, and ≥75%
‘considerable’. Potential small study bias was assessed
by Egger’s test (signicance level P > 0.1) and visual
inspection of funnel plots.
For studies using soy with different amounts of isoa-
vones, we included only the intervention arm using the
lowest isoavone dose. Differences between plant protein
sources were evaluated by subgroup analyses of soy vs.
non-soy interventions, with between-group heteroge-
neity assessed by Cochran’s Q. The meta-analyses were
performed with Stata/SE version 17.0 (StataCorp LLC,
College Station, Texas, USA).
Certainty assessment
We categorized the strength of evidence according to the
World Cancer Research Fund’s grading: ‘Convincing’,
‘Probable’, ‘Limited – suggestive’, ‘Limited – no conclu-
sion’, and ‘Substantial effects unlikely’ (9). The quality
(risk of bias), quantity, consistency, and precision in the
body of evidence were considered in categorizing the
strength of evidence.
Results
Study selection search results
Figure 1 shows the literature search, screening, and the
number of papers/studies excluded (including the rea-
sons) as well as the studies retrieved and included in the
SR. The potentially eligible studies excluded after the
full-text assessment is listed together with reasons in the
online supplement (Supplementary le 2).
Study characteristics
In total, 20 publications were included (Tables 2 and 3).
Out of these, 13 were RCTs (25–37), including between 23
and 140 subjects each (total, n = 906) (Tabl e 2). Seven RCTs
had a crossover design and six a parallel design. Seven of
the RCTs were conducted in USA, three in Germany, two
in Canada, and one in Brazil.
There were seven reports (38–44) from seven cohort
studies, including between 2,332 and 416,104 subjects
(total, n = 720,663 for CVD mortality; n = 5,873 for CHD
incidence; n = 281,341 for T2D incidence) with endpoint
data (Table 3). The cohorts included subjects from USA,
Japan, Finland, and the Netherlands.
Records idenfied through
database searching
(n = 15,090)
ScreeningIncluded EligibilityIdenficaon
Addional records idenfied
through other sources
(n = 1)
Records aer duplicates removed
(n = 9,950)
Records screened
(n = 9,950)
Records excluded
(n = 9,826)
Full-text arcles assessed
for eligibility
(n = 124)
Full-text arcles excluded,
with reasons
RCT with only high
isoflavones n=46
Observaonal study with
parallel design n=22
Wrong exposure n=32
Wrong outcome n=2
Wrong populaon n=1
Duplicate n=1
In total n= 104
Studies included in
qualitave synthesis
(n = 20)
Studies included in
quantave synthesis
(meta-analysis)
(n = 12)
Fig. 1. PRISMA ow diagram for database searches and study screening.
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Animal versus plant-based protein and risk of CVD and T2D
Table 2. Selected characteristics of the randomized controlled studies
Author
Yearcountry
Population Inclusion criteria Design Treatment /
Exposures
Dietary
assessment
methods
Participants
N
Age at inclusion/
start of
intervention
Follow-up
time
Outcomes
Bähr et al.
2013
Germany
Hypercholesterolemic
adults 18–80 years
of age
Total cholesterol concentration of
≥5.2mmol/L at screening
RCT,
Cross-over
25 g protein/day,
5g/100 mL lupin
protein, or 5.1
g/100 mL milk
protein
5-d weighted
food record
66 Group AB: 49.7
(12.8) years
Group BA: 49.4
(13.9) years
8 weeks SBP, DBP
at 8 weeks,
TC, LDL-C,
HDL-C, TG
Bähr et al.
2015
Germany
Hypercholesterolemic
adults 18–80 years
of age
Total cholesterol concentration of
≥5.2mmol/L at screening
RCT,
Cross-over
25 g/d lupin
protein or milk
protein or milk
protein plus 1.6
g/d arginine
3-day food
frequency
protocol
72 (24/
intervention
period)
Group A: 54.0
(9.2) years
Group B: 56.5
(13.2) years
Group C: 59.8
(9.3) years
28 days + 6
weeks wash-
out periods
SBP, DBP,
TC, LDL-C,
HDL-C, TG
Crouse et al.
1999
USA
Subjects with
moderate
hypercholesterolemia
Age 20–70 years with LDL choles-
terol levels between 3.62 mmol/L
(140 mg/dL) and 5.17 mmol/L
(200mg/dL) after following a
run-in diet for 1 month (NCEP
Step I low-fat, low-cholesterol diet
consisting of 30% of energy as fat
(polyunsaturated-monounsaturat-
ed-saturated fat ratio, 1:1:1) and
300 mg of cholesterol daily)
RCT,
Parallell
25 g of soy isolate
or 25 g casein per
day
Isolate soy protein
containing either
3, 27, 37, or 62 mg
isoavones
Three 4-day
records
28 (3 mg
isoavone);
31(casein)
Mean (SD): 52
(11) years
9 weeks TC, LDL-C,
HDL-C, TG.
Primary
comparison
was 62 mg
isoavones
and casein.
Dent et al.
2001
USA
Perimenopausal
women, normocholes-
terolemic, and mildly
hypercholesterolemic
Experiencing ≥10 hot ushes
and/or night sweats per wk, had
irregular menses or cessation of
menses for <1 y, had one or both
ovaries remaining, had a body
mass index (kg/m2) between 19
and 31, were willing to be ran-
domly assigned to treatment, and
were able to participate for 24
wk, follicle-stimulating hormone
concentrations ≥30 iu/L.
RCT,
Parallel
G1) Isoavone-rich
soy protein isolate
G2) Isoavone-
poor soy protein
isolate
G3) Whey protein
40 g/day. Mean pro-
tein intake increased
by 27 g/day.
5-day food
records
24 (G2),
21(G3)
50.2 ± 3.6
(41.9–61.6)
years
24 weeks TC, LDL-C,
HDL-C, TG
Frota et al.
2015
Brazil
Mild or moderate
hypercholesterolemic
adults
Men aged 30–70 years or
postmenopausal women age
45–70 years of age, mild to
moderate hypercholesterolemia
(LDL cholesterol ≥160 mg/dl,
≤190mg/dl).
RCT,
Cross-over
25 g protein in
2 servings of
protein shakes
daily (per cowpea
shake: 12.6pro-
tein, casein shake:
14.1g protein)
24-h dietary
recalls
38 57.0 (SEM 1.7)
years
6 weeks TC, LDL-C,
HDL-C,
TG, glucose
(data not
shown)
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Table 2. (Continued)
Author
Yearcountry
Population Inclusion criteria Design Treatment /
Exposures
Dietary
assessment
methods
Participants
N
Age at inclusion/
start of
intervention
Follow-up
time
Outcomes
Gardner et al.
2001
USA
Postmenopausal,
moderately hypercho-
lesterolemic
women
Postmenopausal (≥1 y since
their last menstrual cycle), were
aged <80 y, and had a body mass
index (BMI; in kg/m2) of 20–31,
LDL-cholesterol concentration
of3.37–4.92 mmol/L), and a
triacylglycerol concentration
<2.82 mmol/L
RCT,
Parallell
42 g/day of soy
protein isolate
(2 × 21 g/d);
Soy-: Isolated
soy protein with
trace amounts
of isoavone;
Soy+: Isolated soy
protein containing
isoavones
Milk protein
3 day food
records
30, 33 Milk: 57.7 (6.0)
yrs, Soy-: 58.4
(7.2) yrs, Soy+:
62.6 (7.3) yrs
12 weeks TC, LDL-C,
HDL-C, TG
Gardner et al
2007
USA
Hypercholesterolemic
adults
LDL-C concentration 4.14–5.69
mmol/L and Framingham risk
score of ≤10% based on gender,
age, LDL-C, HDL-C, blood pres-
sure, and diabetes
RCT,
Cross
over
25 g protein from
either milk type
(32 oz whole bean
soy drink, 28 oz
soy protein isolate
drink, 18.5oz dairy
milk). Isoavones:
125 mg in whole
bean soy milk, 39
mg in SPI drink)
Milk con-
sumption
logs and
3-day food
records
28 52 (9) years 4 weeks TC, LDL-C,
HDL-C,
TG, fasting
insulin AUC,
fasting
glucose
Jenkins et al.
2010
Canada
Hypercholesterolemic
adults
Men >21 y or postmenopausal
women with LDL-C >3.5 mmol/L
RCT,
Cross-over
30 g barley or
casein protein per
2,000 kcal daily
(18–19 g protein
per 100 g)
7-day dietary
history
23 56 (2) (range
41–69) years
6 weeks TC, TG,
LDL-C,
HDL-C, SBP,
DBP
Lichtenstein
et al.
2002
USA
Moderately
hypercholesterolemic
Men and women
>50 years with LDL cholesterol
levels greater than 3.36 mmol/L;
and postmenopausal (for women)
RCT,
Cross-over
Soy/-: Soy protein
depleted of isoa-
vones, Soy/+: Soy
protein enriched in
isoavones
Animal/-: Animal
protein with-
out isoavones,
Animal/+: Animal
protein enriched in
isoavones
2/3 of total protein
intake (i.e., 10 E%, 25
g protein/4.2 MJ).
Not
provided
42 62.7 (8.8) years 6 weeks TC, LDL-C,
HDL-C, TG
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Animal versus plant-based protein and risk of CVD and T2D
Table 2. (Continued)
Author
Yearcountry
Population Inclusion criteria Design Treatment /
Exposures
Dietary
assessment
methods
Participants
N
Age at inclusion/
start of
intervention
Follow-up
time
Outcomes
McVeigh et al.
2006
Canada
Healthy young males Healthy males between the ages
of 20 and 40 y and with a body
mass index (BMI; in kg/m2) of
19–29.
RCT,
Parallel
Low-iso SPI: Low
isoavone soy
protein isolate;
High-iso SPI: High
isoavone soy
protein isolate
According to
body weight.
High-iso: 0.75 mg
isoavones/kg/d.
MPI: Milk protein
isolate
3-d food
record
70 (35; 35) 27.9 (5.7) years 57 days TC, LDL-C,
HDL-C, TG
Santo et al.
2008
USA
Healthy, young, seden-
tary males
Healthy, male, age 18–30 y, nor-
mocholesterolemic, BMI between
18 and 26 kg/m2
RCT,
Parallel
Soy-: Isoavone-
poor soy protein
isolate; Soy+:
Isoavone-rich soy
protein isolate
Milk: Milk protein
isolate
25 g protein/day
3-day food
records
30 (11; 10; 9) Milk: 24.0 (0.9)
years, Soy-:
23.6 (0.5) years,
Soy+: 25.1 (0.8)
years
28 days TC, LDL-C,
HDL-C, TG,
Glucose
Steinberg
et al
2003
USA
Healthy, postmeno-
pausal women
Menopausal status, as dened
by the absence of menstrual
bleeding in the past 12 mo and
follicle-stimulating hormone
concentrations of ≥ 23 IU/L
RCT,
Cross-over
Soy-: Isolated soy
protein with trace
amounts of isoa-
vones; Soy+: Isolated
soy protein with
naturally occurring
isoavones
TMP: Total milk
protein
25 g protein/day
3-day food
records
28 54.9 (1.0) years 6 weeks TC, LDL-C,
HDL-C, TG
Weiße et al.
2010
Germany
Moderate, hypercho-
lesterolemic adults
21 to 70 years of age with
moderate hypercholesterolemia
(5.7–7.9 mM)
RCT,
Parallel
Lupin protein
Casein protein
35 g protein/day
Diaries 43 (22; 21) 43.9 (11.8)
years
6 weeks TC, LDL-C,
HDL-C, TG,
Glucose
SBP, systolic blood pressure; DBP, diastolic blood pressure; TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; TG, triacylglycerol; BMI, body
mass index; RCT, randomized controlled trial; SD, standard deviation.
Table 2. (Continued)
Author
Yearcountry
Population Inclusion criteria Design Treatment /
Exposures
Dietary
assessment
methods
Participants
N
Age at inclusion/
start of
intervention
Follow-up
time
Outcomes
Gardner et al.
2001
USA
Postmenopausal,
moderately hypercho-
lesterolemic
women
Postmenopausal (≥1 y since
their last menstrual cycle), were
aged <80 y, and had a body mass
index (BMI; in kg/m2) of 20–31,
LDL-cholesterol concentration
of3.37–4.92 mmol/L), and a
triacylglycerol concentration
<2.82 mmol/L
RCT,
Parallell
42 g/day of soy
protein isolate
(2 × 21 g/d);
Soy-: Isolated
soy protein with
trace amounts
of isoavone;
Soy+: Isolated soy
protein containing
isoavones
Milk protein
3 day food
records
30, 33 Milk: 57.7 (6.0)
yrs, Soy-: 58.4
(7.2) yrs, Soy+:
62.6 (7.3) yrs
12 weeks TC, LDL-C,
HDL-C, TG
Gardner et al
2007
USA
Hypercholesterolemic
adults
LDL-C concentration 4.14–5.69
mmol/L and Framingham risk
score of ≤10% based on gender,
age, LDL-C, HDL-C, blood pres-
sure, and diabetes
RCT,
Cross
over
25 g protein from
either milk type
(32 oz whole bean
soy drink, 28 oz
soy protein isolate
drink, 18.5oz dairy
milk). Isoavones:
125 mg in whole
bean soy milk, 39
mg in SPI drink)
Milk con-
sumption
logs and
3-day food
records
28 52 (9) years 4 weeks TC, LDL-C,
HDL-C,
TG, fasting
insulin AUC,
fasting
glucose
Jenkins et al.
2010
Canada
Hypercholesterolemic
adults
Men >21 y or postmenopausal
women with LDL-C >3.5 mmol/L
RCT,
Cross-over
30 g barley or
casein protein per
2,000 kcal daily
(18–19 g protein
per 100 g)
7-day dietary
history
23 56 (2) (range
41–69) years
6 weeks TC, TG,
LDL-C,
HDL-C, SBP,
DBP
Lichtenstein
et al.
2002
USA
Moderately
hypercholesterolemic
Men and women
>50 years with LDL cholesterol
levels greater than 3.36 mmol/L;
and postmenopausal (for women)
RCT,
Cross-over
Soy/-: Soy protein
depleted of isoa-
vones, Soy/+: Soy
protein enriched in
isoavones
Animal/-: Animal
protein with-
out isoavones,
Animal/+: Animal
protein enriched in
isoavones
2/3 of total protein
intake (i.e., 10 E%, 25
g protein/4.2 MJ).
Not
provided
42 62.7 (8.8) years 6 weeks TC, LDL-C,
HDL-C, TG
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Table 3. Selected characteristics of the cohort studies
Author
Yearcountry
Population Design Dietary assessment
methods
Number
recruited
Number
analyzed
Age at inclusion/start
ofintervention
Follow-up time Outcomes
Budhathoki et al.
2019
Japan
Eleven public health center areas
across Japan, included in the
Japan Public Health Center-based
Prospective Cohort (JPHC) Study
Residents aged 40 to 69 years
PC 138-item semiquan-
titative FFQ
140,420 (61,595
from cohort 1
and 78,825 from
cohort 2)
70,696 Men: mean (SD): 55.6
(7.6) years, Women:
mean (SD): 55.8 (7.7)
years
Mean 18 years CVD mortality
Huang et al.
2020
USA
National Institutes of Health-American
Association of Retired Persons (NIH-
AARP) Diet and Health Study
Adults 50–71 years
PC National
Cancer Institute
Diet History
Questionnaire
(DHQ) of 124
dietary items (FFQ)
566,398 416,104 Median (SD) ages: Men:
62.2≈(5.4) years, Women:
62.0 (5.4) years
16 years (median,
15.5 years; IQR,
15.5–15.8),
6,009,748 person
years
CVD mortality
Malik et al. 2016
USA
Nurses’ Health Study (NHS), Nurses’
Health Study II (NHS II), and Health
Professionals Follow-up Study (HPFS)
Female registered nurses and male
health professionals
PC 131-item FFQ 289,900 (NHS:
121,700, NHS II:
116,671, HPFS:
51,529)
205,802 (NHS:
72,992, NHS II:
92,088, HPFS:
40,722)
NHS: mean: ≈50.1
(30–55)years
NHS II: mean: ≈36.0
(24–42)years
HPFS: mean: ≈53.0
(40–75)years
4,146,216 per-
son-years (18–24
years)
T2D incidence
Song et al.
2016
USA
Nurses’ Health Study (NHS) and
Health Professionals Follow-up Study
(HPFS)
Female registered nurses and male
health professionals
PC 131-item FFQ 173,229 (NHS:
121,700, HPFS:
51,529)
131,342 (NHS:
85,013, HPFS:
46,329)
NHS: 30–55 years,
HPFS:40–75 years
3,540,791
person-years
CVD mortality
Sun et al.
2021
USA
Women’s Health Initiative (WHI)
Postmenopausal women
PC 122-item FFQ 137,481 (OS:
90,009; CT:
47,472)
102,521 (OS:
63,593; CT:
38,928)
50–79 years 1,876,205 per-
son-years (18.1
years on average)
CVD mortality
Virtanen et al.
2017
Finland
Kuopio Ischaemic Heart Disease Risk
Factor Study (KIHD)
Middle-aged and older Finnish men
PC 4-d food record 2,682 2,332 42–60 years Mean: 19.3 years T2D incidence
Voortman et al
2021
The Netherlands
Participants from the Rotterdam Study,
3 different cohorts (RI, RII, RIII)
Population based cohort study
PC 170-item semi-
quantitative FFQ;
RS-III, more compre-
hensive FFQ con-
taining 389 items
14,926 com-
plete dietary
data from 9,701
participants
5,873 Mean age 61.6 (7.9)
(60.8%females)
74,776
person-years
CHD incidence
FFQ, food frequency questionnaire; PC, prospective cohort; CHD, coronary heart disease; CVD, cardiovascular disease; T2D, type 2 diabetes.
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Animal versus plant-based protein and risk of CVD and T2D
Types of intervention/exposures
Eight RCTs compared the effect of low-isoavone soy
protein supplementation to casein or milk protein supple-
mentation on different outcomes (27, 28, 30, 31, 33–36)
(Table 2). Three RCTs (25, 26, 37) compared the effect of
lupin protein supplementation to milk protein or casein
supplementation, and one (27) compared, in addition, the
effect of lupin protein supplementation to milk protein
+ arginine supplementation on different outcomes. One
RCT investigated the effect of barley protein supplemen-
tation in comparison to casein supplementation in bread
(32), and one compared the effect of cowpea protein sup-
plementation to casein supplementation (29) on different
outcomes. The protein supplementation amount ranged
between 25 and 30 mg/d for all studied protein sources.
The outcomes in all studies were related to lipid metabo-
lism. In some RCTs, the effects on glucose metabolism or
blood pressure were studied.
Four reports from ve prospective cohorts investi-
gated the association between plant protein as E% sub-
stitution of animal protein and risk of CVD mortality
(38, 39, 41, 42) (Table 3) and one on CHD incidence (44).
Two reports from four prospective cohorts examined the
association with plant protein intake as E% substitution
of animal protein and the incidence of T2D (40, 43)
(Table 3).
Outcome assessment
The duration of the interventions in the RCTs ranged from
4 weeks to 24 weeks, all reporting on serum/plasma total
cholesterol concentrations (total cholesterol), serum/plasma
LDL (low-density lipoprotein)-cholesterol concentrations
(LDL-cholesterol), serum/plasma HDL (high-density lipo-
protein)-cholesterol concentrations (HDL-cholesterol), and-
serum/plasma triacylglycerol concentrations (triacylglycerol,
TG). In addition, three studies (25, 26, 32) reported on effects
on blood pressure, one on fasting serum/plasma insulin con-
centration (30), and four on blood glucose concentration
(29, 30, 35, 37). If blood was drawn at several time points,
only the results from the baseline and latest time point were
considered. In the cohort studies, the follow-up time between
assessment of diet and outcome ranged from 16 to 19.3 years
(median or average in those where it was reported).
Risk of bias in included studies
The risk of bias assessment per domain in RCT studies is
outlined in Figs. 2 and 3. Five RCTs had overall low concerns
for risk of bias (25–27, 30, 31). Four RCTs had overall some
concerns, due to the lack of information on the randomiza-
tion process (28, 29, 34, 37). Four RCTs had overall high
concern of bias, mostly due to non-adherence to the study
intervention (32, 33, 35, 36). The risk of bias for all prospec-
tive cohort studies was moderate overall (Figs. 4 and 5).
Fig. 2. Risk of bias per domain and overall, for all included RCT studies. RCT, randomized controlled trials.
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Plant proteins and blood lipids
The effect on total cholesterol, LDL-cholesterol, HDL-
cholesterol, or triacylglycerol of soy protein in com-
parison to animal protein sources was studied in eight
RCTs (27, 28, 30, 31, 33–36), of which three were cross-
over studies (Tables 2 and 4). Three studies (25, 26, 37)
explored the effect of lupin protein on blood lipids in
hypercholesterolemic subjects, one studied the effect
of barley protein (32), and one of cow-pea protein (29)
(Tables 3 and 4).
Both crossover and parallel studies were pooled in the
meta-analyses. The summary effect sizes showed signi-
cantly decreased total cholesterol (Fig. 6; -0.11 mmol/L,
95% CI, -0.22, -0.01, I2 = 8.3%) and LDL-cholesterol
(Fig.7; -0.14 mmol/L, 95% CI, -0.25, -0.02, I2 = 43.8%),
with plant protein interventions compared to animal
protein, a borderline signicantly increased HDL-
cholesterol (Fig. 8; 0.04 mmol/L, 95% CI, 0.00, 0.07, I2
= 0.01%), but unsignicant effects on TG (Fig. 9; -0.00
mmol/L, 95% CI, -010, 0.09, I2 = 0.00%). It should be
Fig. 3. Summary of bias per domain and overall, for all included RCT studies. RCT, randomized controlled trials.
Fig. 4. Risk of bias per domain and overall, for all included cohort studies.
Fig. 5. Summary risk of bias per domain and overall, for all included cohort studies.
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Animal versus plant-based protein and risk of CVD and T2D
Table 4. Summary of ndings in randomized controlled trials
Author, year Plant protein outcomes Animal protein outcomes Comparison between
groups (P-value)
Summary of
resultsa
Risk of
bias
Soy
Crouse et al. 1999 Soy, 3 mg isoavones
Mean (SD) at 9 weeks:
TC: 6.10 (0.65) mmol/L
LDL: 4.14 (0.57) mmol/L
HDL: 1.19 (0.28) mmol/L
TG: 1.72 (0.65) mmol/L
Casein
Mean (SD) at 9 weeks:
TC: 6.23 (0.70) mmol/L
LDL: 4.27 (0.59) mmol/L
HDL: 1.14 (0.23) mmol/L
TG: 1.89 (0.84) mmol/l
TC: P = NS
LDL: P = NS
HDL: P = NS
TG: P = NS
TC: ↔
LDL: ↔
HDL: ↔
TG: ↔
Low
Dent et al. 2001 SPI- = soy protein (low
isoavones)
Estimated values from Fig. 1:
Mean at 24 weeks:
TC: 5.47 mmol/L
LDL: 3.51 mmol/L
Median:
HDL: 1.07 mmol/L
TG: 1.07 mmol/L
Whey protein
Estimated values from
Fig. 1:
Mean at 24 weeks:
TC: 5.46 mmol/L
LDL: 3.52 mmol/L
Median: HDL: 1.40 mmol/L
TG: 1.35 mmol/L
TC: 0.96
LDL: 0.76
HDL: 0.99
TG: 0.9
TC: ↔
LDL: ↔
HDL: ↔
TG: ↔
Some
Gardner et al. 2001 Soy-
Mean (SD) at 12 weeks:
TC: 5.9 (0.9) mmol/L
LDL: 3.8 (0.8) mmol/L
HDL: 1.5 (0.2) mmol/L
TG: 1.3 (0.6) mmol/L
Milk
Mean (SD) at 12 weeks:
TC: 5.9 (0.7) mmol/L
LDL: 3.7 (0.6) mmol/L
HDL: 1.5 (0.4) mmol/L
TG: 1.4 (1.0) mmol/L
TC: n.s. between soy and
milk
LDL: n.s. between soy
andmilk
HDL: 1.0
TG: 0.3
TC: ↔
LDL: ↔
HDL: ↔
TG: ↔
Low
Gardner et al. 2007 Mean (SD) at 4 weeks:
LDL:
Whole bean Soy milk: 4.17
(0.52) mmol/L
Soy protein isolate milk: 4.17
(0.67) mmol/L
Insulin AUC:
Whole bean Soy milk: 44 (20)
Soy protein isolate milk: 45 (25)
Glucose. fasting:
Whole beans milk: 5.2 (0.5)
mmol/L
Soy protein isolate milk: 5.1 (0.6)
mmol/L
Dairy milk
Mean (SD) at 4 weeks:
LDL: 4.39 (0.62) mmol/L
Insulin AUC: 44 (24)
Glucose. fasting: 5.1 (0.6)
mmol/L
Both soy milks vs. Dairy
milk:
LDL: P = 0.02
HDL: P = 0.8
TG: P = 0.4
Insulin: 0.9
Glucose: 0.4
LDL: ↓
HDL: ↔
TG: ↔
Insulin: ↔
Glucose: ↔
Low
Lichtenstein
et al.
2002
Soy-
Mean (SD) at 6 weeks:
TC: 6.37 (1.12) mmol/L
LDL: 4.34 (0.92) mmol/L
HDL: 1.36 (0.34) mmol/L
TG: 1.27 (0.50) mmol/L
Animal protein
Mean (SD) at 6 weeks:
TC: 6.47 (1.17) mmol/L
LDL: 4.42 (0.97) mmol/L
HDL: 1.33 (0.32) mmol/L
TG: 1.44 (0.57) mmol/L
Between proteins:
TC: P = 0.017.
LDL: P = 0.042.
HDL: P = 0.034.
TG: P < 0.0001.
Between
proteins:
TC: ↓
LDL: ↓
HDL:
TG: ↓
High
McVeigh et al. 2006 Low-iso Soy protein
Least-squares mean (SE) at
57days:
TC: 4.47 (0.06) mmol/L
LDL: 2.71 (0.05) mmol/L
HDL: 1.15 (0.02) mmol/L
TG: 1.35 (0.07) mmol/L
Milk protein
Least-squares mean (SE) at
57 days:
TC: 4.55 (0.06) mmol/L
LDL: 2.86 (0.05) mmol/L
HDL: 1.10 (0.02) mmol/L
TG: 1.30 (0.07) mmol/L
TC: n.s.
LDL: n.s.
HDL: n.s.
Non-HDL: n.s.
TG: n.s.
TC: ↔
LDL: ↔ (↓
in equol
excretors)
HDL: ↔
Non-HDL: ↔
TG: ↔
Some
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Table 4. (Continued)
Author, year Plant protein outcomes Animal protein outcomes Comparison between
groups (P-value)
Summary of
resultsa
Risk of
bias
Santo et al.
2000
Low-isoavone soy protein
Mean (SEM) at 28 days:
TC: 4.91 (0.34) mmol/L
LDL: 2.92 (0.38) mmol/L
HDL: 1.32 (0.11) mmol/L
TG: 1.42 (0.27) mmol/L
Glucose: 5.3 (0.2) mmol/L
Milk protein
Mean (SEM) at 28 days:
TC: 4.27 (0.25) mmol/L
LDL: 2.66 (0.32) mmol/L
HDL: 1.19 (0.15) mmol/L
TG: 1.04 (0.18) mmol/L
Glucose: 5.4 (0.3) mmol/L
Low-isoavone soy vs.
Milk: No differences
TC: ↔
LDL: ↔
HDL: ↔
TG: ↔
Glucose: ↔
High
Steinberg et al. 2003 Soy-
Mean (SEM) at 6 weeks:
TC: 4.92 (0.2) mmol/L
LDL: 2.87 ± 0.1 mmol/L
HDL: 1.55 ± 0.1 mmol/L
TG: 1.08 ± 0.1 mmol/L
Change from baseline:
TC: 0.01 mmol/l
LDL: -0.02 mmol/l
Milk protein
Mean (SEM) at 6 weeks:
TC: 5.00 ± 0.1 mmol/L
LDL: 2.94 ± 0.1 mmol/L
HDL: 1.61 ± 0.1 mmol/L
TG: 0.98 ± 0.1 mmol/L
Change from baseline
TC: +0.08 mmol/l
LDL: +0.04 mmol/l
All values non-signicant
between diets
TC: ↔
LDL: ↔
HDL: ↔
TG: ↔
High
Lupin
Bähr et al.
2013
Lupin
Change from baseline (mean
(SD)) to 8 weeks:
TC: 0.05 (0.44) mmol/L
LDL: 0.08 (0.50) mmol/l
HDL: -0.05 (0.19) mmol/L
TG: 0.19 (0.45) mmol/L
SBP/DBP: -8.4 (13.6)/ -2.7 (7.5)
mmHg
Casein
Change from baseline
(mean (SD)) to 8 weeks:
TC: 0.02 (0.49) mmol/L
LDL: -0.06 (0.34) mmol/L
HDL: -0.02 (0.13) mmol/L
TG: 0.16 (0.77) mmol/L
SBP/DBP: -5.9 (12.9)/ -1.5
(7.7) mmHg
TC: P = 0.52
LDL: P = 0.90
HDL: P = 0.20
TG: P = 0.77
SBP/DBP: P = 0.29/0.31
TC: ↔
LDL: ↔
HDL: ↔
(↑ at 4 weeks)
TG: ↔
SBP: ↔
DBP: ↔
Low
Bähr et al.
2015
Lupin
Mean (SD) at 4 weeks:
TC: 6.13 (0.95) mmol/L
LDL: 4.01 (0.87) mmol/L
HDL: 1.35 (0.37) mmol/L
TG: 1.69 (1.29) mmol/L
SBP/DBP: 142.2 (20.8) / 87.0
(9.9) mmHg
Milk protein
Mean (SD) at 4 weeks:
TC: 6.23 (0.97) mmol/L
LDL: 4.08 (0.95) mmol/L
HDL: 1.36 (0.35) mmol/L
TG: 1.77 (1.42) mmol/L
SBP/DBP: 140.3 (19.2) /
86.8 (9.8) mm Hg
TC: P = 0.07
LDL: P = 0.044
HDL: P = 0.37
TG: P = 0.49
SBP/DBP: P = 0.35/0.84
TC: ↔ (P =
0.07)
LDL: ↓
HDL: ↔
TG: ↔
SBP: ↔
DBP: ↔
Low
Weiße et al.
2010
Lupin protein
Mean (SD) at 6 weeks:
TC: 5.17 (0.59) mmol/L
LDL: 3.30 (0.64) mmol/L
HDL: 1.67 (0.42) mmol/L
TG: 1.32 (0.72) mmol/L
Glucose: 5.10 (0.75) mmol/L
Casein
Mean (SD) at 6 weeks:
TC: 5.32 (0.77) mmol/L
LDL: 3.50 (0.73) mmol/L
HDL: 1.54 (0.35) mmol/L
TG: 1.26 (0.70) mmol/L
Glucose: 5.14 (0.78)
mmol/L
At 6 weeks
TC: P = 0.509
LDL: P = 0.380
HDL: P = 0.294
TG, P = 0.715
Glucose: P = 0.861
Difference in change:
TC: P = 0.9
LDL: P = 0.384
HDL: P = 0.150
TG: P = 0.068
Glucose: P = 0.992
Between
groups, at 6
weeks
TC: ↔
LDL: ↔
HDL: ↔
TG: ↔
Glucose: ↔
Difference in
change:
TC: ↔
LDL: ↔
HDL: ↔
TG: ↔ (P =
0.068)
Some
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Animal versus plant-based protein and risk of CVD and T2D
noted that Dent et al. (28) could not be meta-analyzed
as results were only presented as P-values, and Gardner
et al. (30) could only be included in the LDL-cholesterol
meta-analysis.
In subsequent assessment, the meta-analyses of the
RCTs were stratied by the plant protein source with
subgroup analyses of soy vs. non-soy interventions
(Supplementary le 3). No clear differences in blood lip-
ids between the soy and the non-soy interventions in com-
parison to animal protein were observed.
Based on inspection of funnel plots (not shown), and
Egger’s test for all meta-analyses including all interven-
tion studies, we did not nd evidence of publication bias
in the form of small study-effects bias.
Table 4. (Continued)
Author, year Plant protein outcomes Animal protein outcomes Comparison between
groups (P-value)
Summary of
resultsa
Risk of
bias
Cowpea
Frota et al. 2015 Cowpea
Mean (SEM) at 6 weeks:
TC: 6.0 (0.11) mmol/L
LDL: 3.67 (0.09) mmol/L
HDL: 1.48 (0.04) mmol/L
TG: 1.84 (0.16) mmol/L
Casein
Mean (SEM) at 6 weeks:
TC: 6.58 (0.12) mmol/L
LDL: 4.26 (0.09) mmol/L
HDL: 1.41 (0.04) mmol/L
TG: 1.95 (0.25) mmol/L
Percentage changes
TC: P < 0.001
LDL: P < 0.001
HDL: P = 0.044
TG: –
TC: ↓
LDL: ↓
HDL: ↑
TG: ↔
Some
Barley
Jenkins et al. 2010 Barley
Mean (SEM) at 4 weeks:
TC: 5.9 (0.19) mmol/L
LDL: 3.95 (0.16) mmol/L
HDL: 1.30 (0.06) mmol/L
TG: 1.42 (0.11) mmol/L
Blood pressure
SBP: 118 (2) mmHg
DBP: 69 (2) mmHg
Casein
Mean (SEM) at 4 weeks:
TC: 5.79 (0.19) mmol/L
LDL: 3.93 (0.18) mmol/L
HDL: 1.27 (0.06) mmol/L
TG: 1.32 (0.10) mmol/L
Blood pressure
SBP: 118 (3) mmHg
DBP: 69 (2) mmHg
Difference between
treatments
TC: P = 0.57
LDL: P = 0.896
HDL: P = 0.184
TG: P = 0.334
Blood pressure
SBP, P = 0.639
DBP, P = 0.418
TC: ↔
LDL: ↔
HDL: ↔
TG: ↔
SBP: ↔
DBP: ↔
High
SBP, systolic blood pressure; DBP, diastolic blood pressure; TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density
lipoprotein cholesterol; TG, triacylglycerol; AUC, area under curve; SE, standard error of mean; SD, standard deviation. aArrows indicate the direc-
tion of association.
Fig. 6. Meta-analysis of RCT studies of total cholesterol. Forest plot showing mean differences with 95% CI in total choles-
terol (mmol/l) by replacing animal protein with plant protein. The summary effect estimate (white diamond) was estimated by a
restricted maximum likelihood random-effects model. RCT, randomized controlled trials; CT, condence interval.
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Plant protein, blood pressure, blood glucose, and insulin
concentration
Two studies (25, 26) investigated the impact of lupin pro-
tein or barley (32) and observed no effect on blood pressure
compared to the animal protein (Tables 2 and 4). Three
papers studied the effect of plant protein in comparison
with animal protein on blood glucose (30, 35, 37) and one
on fasting insulin (30), with no differences between the
treatment groups. No meta-analyses were conducted for
these outcomes, as the number of studies were insufcient.
Fig. 7. Meta-analysis of RCT studies of LDL-cholesterol. Forest plot showing mean differences with 95% CI in total choles-
terol (mmol/l) by replacing animal protein with plant protein. The summary effect estimate (white diamond) was estimated by a
restricted maximum likelihood random-effects model. RCT, randomized controlled trials; CT, condence interval.
Fig. 8. Meta-analysis of RCT studies of HDL-cholesterol. Forest plot showing mean differences with 95% CI in total choles-
terol (mmol/l) by replacing animal protein with plant protein. The summary effect estimate (white diamond) was estimated by a
restricted maximum likelihood random-effects model. RCT, randomized controlled trials; CT, condence interval.
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Animal versus plant-based protein and risk of CVD and T2D
Substitution of animal protein with plant protein and CVD
Only one prospective cohort study (44) was retrieved
that focused on the incidence of CHD using substitution
model design (Tables 3 and 5). Although non-signicant,
a higher plant protein intake tended to be associated with
a lower risk of CHD when consumed at the expense of
animal protein. All four prospective studies (38, 39, 41,
42) with an isocaloric substitution of animal protein
with plant protein showed lower risk of CVD mortality
(Tables 3 and 5). Of these, Song et al. (41) found that sub-
stituting animal protein from processed or unprocessed
red meat, sh, or dairy with plant protein was associated
with lower CVD mortality. Budhathoki et al. (38) found
that replacing animal protein from red meat (not from
processed meat, chicken, egg, dairy, or sh) with plant
protein reduced CVD mortality. Huang et al. (39) found
that replacing total animal protein with plant protein was
associated with lower mortality from CVD, heart disease,
and stroke in both men and women. When separating on
sources of animal protein, results remained for red meat,
dairy, and egg, but replacing white meat protein with
plant protein was only signicantly associated with lower
stroke mortality in men.
Substitution of animal protein with plant protein and T2D
incidence
Only two papers (40, 43) fullled our inclusion crite-
ria for T2D incidence, and both showed associations
with reduced T2D incidence with isocaloric substitution
of animal protein with plant protein (Tables 3 and 5).
Virtanen et al. (43) also showed that replacing any ani-
mal protein except for protein from eggs with energy from
plant protein was associated with a 14–20% decreased risk
of T2D, although not all associations reached statistical
signicance.
Certainty in the evidence
The evidence for a favorable association between plant pro-
tein intake in comparison to animal protein and CVD mor-
tality was considered limited-suggestive based on consistent
results from cohort studies with moderate risk of bias, sup-
ported by evidence of biological plausibility from the RCTs.
The corresponding evidence for T2D incidence was consid-
ered limited, suggestive, while the few available RCT studies
on blood glucose and insulin did not support an effect.
Discussion
Summary of key ndings
This SR summarizes both RCTs and cohort studies for
whether substituting plant protein for animal protein is
associated with lower risk of CVD and T2D or lower
levels of cardiometabolic risk factors. While the cohort
studies reported associations with decreased risks of
CVD and T2D in substitution models of animal protein
with plant protein, the biological plausibility based on
the RCTs was supported for CVD alone. Evidence was
considered limited-suggestive for reduced CVD mortal-
ity and T2D, when replacing animal protein with plant
protein.
Fig. 9. Meta-analysis of RCT studies of triacylglycerol. Forest plot showing mean differences with 95% CI in total cholesterol
(mmol/l) by replacing animal protein with plant protein. The summary effect estimate (white diamond) was estimated by a
restricted maximum likelihood random-effects model. RCT, randomized controlled trials; CT, condence interval.
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Table 5. Summary of ndings from cohort studies
Author
Year
Population
Outcome Exposure Substitution of animal protein with
plant protein
Conclusions Overall risk of
bias
CVD
Budhathoki
etal. 2019
Japan
CVD, heart
disease and
cerebrovas-
cular disease
mortality
Animal protein, plant protein;
Mean (SD) intakes, expressed as
percentage of total energy:
Animal protein: 7.7 (2.7) Plant
protein: 6.7 (1.4)
Substituting 3 E% plant protein
for animal protein:
HR (95% CI)
Red meat: 0.58 (0.38–0.86)
Processed meat: 0.58 (0.29–1.14)
Chicken: 0.84 (0.50–1.42)
Egg: 0.79 (0.57–1.11)
Dairy: 0.82 (0.56–1.18)
Fish: 0.86 (0.69–1.08)
Replacement of
red or processed
meat protein with
plant protein was
associated with a
decreased risk of
total, cancer-related,
and CVD-related
mortality. The study
suggests that encour-
aging diets with higher
plant-based protein
intake may contribute
to long-term health
and longevity.
Moderate
Huang et al.
2020
USA
CVD, heart
disease
and stroke
mortality
Median plant protein intake:
Men: 26.9 g/d (14.4 g/1,000
kcal/d)
Women: 21.6 g/d (14.9 g/1,000
kcal/d)
Substituting 3 E% plant protein
for animal protein
HR (95% CI)
Total animal protein
CVD
Men: 0.89 (0.85–0.94)
Women: 0.88 (0.82–0.94)
Heart disease:
Men: 0.91 (0.86–0.96)
Women: 0.91 0.90 (0.84–0.98)
Stroke
Men: 0.78 (0.68–0.90)
Women: 0.81 (0.70–0.94)
Red meat protein
CVD
Men: 0.88 (0.83–0.93)
Women: 0.82 (0.76–0.89)
Heart disease
Men: 0.89 (0.84–0.94)
Women: 0.84 (0.77–0.92)
Stroke
Men: 0.79 (0.68–0.91)
Women: 0.79 0.75 (0.63–0.89)
White meat protein
CVD
Men: 0.95 (0.90–1.01)
Women: 0.94 (0.87–1.02)
Heart disease
Men: 0.97 (0.91–1.03)
Women: 0.97 (0.89–1.05)
Stroke
Men: 0.83 (0.71–0.96)
Women: 0.90 (0.76–1.06)
Small but signicant
associations between
higher intake of
plant protein and
lower overall and
CVD mortality, with
prominent inverse
associations observed
for replacement of
egg protein and red
meat protein with
plant protein.
Moderate
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Animal versus plant-based protein and risk of CVD and T2D
Table 5. (Continued)
Author
Year
Population
Outcome Exposure Substitution of animal protein with
plant protein
Conclusions Overall risk of
bias
Dairy protein
CVD
Men: 0.89 (0.84–0.94)
Women: 0.88 (0.82–0.95)
Heart disease
Men: 0.91 (0.86–0.97)
Women: 0.92 (0.84–0.99)
Stroke
Men: 0.77 (0.66–0.89)
Women: 0.80 (0.69–0.94)
Egg protein
CVD
Men: 0.74 (0.67–0.82)
Women: 0.72 (0.63–0.83)
Heart disease
Men: 0.76 (0.69–0.85)
Women: 0.72 (0.62–0.85)
Stroke
Men: 0.67 (0.52–0.88)
Women: 0.75 (0.55–1.03)
Song et al.
2016
USA
CVD
mortality
Animal protein, plant protein;
‘Percentage of total energy:
Animal protein: 14%,
Plant protein: 4%’
Replacement of 3% energy from
various animal protein sources
with plant protein
HR (95% CI)
Processed red meat: 0.61
(0.48–0.78)
Unprocessed red meat: 0.83
(0.76–0.91)
Poultry: 0.91 (0.83–1.00)
Fish: 0.88 (0.80–0.97)
Egg: 0.88 (0.75–1.04)
Dairy: 0.89 (0.80–0.98)
Substitution of plant
protein for animal
protein, especially
from processed red
meat, may confer a
substantial health
benet.
Moderate
Sun et al.
2021
USA
CVD
mortality
Animal protein, plant protein
Median percentage of total
energy: Animal protein: 7.5%
Plant protein: 3.5%
Replacement of 5% of energy
from animal protein with plant
protein
HR (95% CI)
CVD: 0.81 (0.72–0.92) (estimated
from gure)
Substitution of animal
protein with plant
protein was associ-
ated with lower CVD
mortality.
Moderate
Voortman
et al. 2021
CHD
incidence
Total protein, animal protein, and
plant protein
Mean (SD)
Total protein in g/d 85.4 (23.9)
Total protein in E% 16.3 (2.9)
Plant protein in g/d 32.3 (11.9)
Plant protein in E% 6.1 (1.3)
Animal protein in g/d 53.0 (18.3)
Animal protein in E% 10.2 (3.1)
Replacement of 5% energy
intake from animal protein
with plant protein (and other
macronutrients)
HR (95% CI)
0.69 (0.38–1.23)
Macronutrient
composition was not
signicantly associated
with CHD incidence
or cardiometabolic
risk factors.
Moderate
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Table 5. (Continued)
Author
Year
Population
Outcome Exposure Substitution of animal protein with
plant protein
Conclusions Overall risk of
bias
T2D
Virtanen
et al.
2017
Finland
Incident T2D Total protein, animal protein, and
plant protein
Mean (SD) g/day:
Total: 92.9 (14.4)
Animal: 64.8 (15.4)
Vegetable: 25.8 (6.0)
Replacement of 1% energy from
different animal protein sources with
plant protein:
HR (95% CI)
Animal protein: 0.81 (0.67–0.98)
Total meat protein: 0.83
(0.68–1.01)
Red meat: 0.82 (0.67–1.01)
Processed red meat: 0.80
(0.64–0.99)
Unprocessed red meat: 0.83
(0.68–1.01)
Fish: 0.85 (0.69–1.04)
Dairy: 0.79 (0.65–0.97),
Non-fermented dairy: 0.79
(0.64–0.97)
Fermented dairy: 0.79 (0.65–0.97)
Egg: 1.11 (0.68–1.82)
Favoring protein
from plant sources
and eggs over other
animal sources may
be benecial in the
prevention of T2D.
Moderate
Malik et al.
2016
USA
Incident T2D Total protein, animal protein,
and plant protein
Mean percentages of energy
intake:
NHS:
Total protein: 18.1%
Animal protein: 15.1%
Vegetable protein: 5%
NHS II:
Total: 18.9%
Animal: 13.7%
Vegetable protein: 7.3%
HPFS:
Total: 18.2%
Animal: 13.0%
Vegetable protein: 5.1%
Substitution of vegetable protein
for animal protein:
HR (95% CI)
0.77 (0.70–0.84)
Substituting vegetable
protein for animal
protein was associ-
ated with reduced
risk of T2D.
Moderate
CHD, coronary heart disease; CVD, cardiovascular disease; T2D, type 2 diabetes; HR, hazard ratio; CI, condence interval.
Strengths and limitation of review
A strength of this review is that we followed established
processes for undertaking robust SRs. The NNR 2022
Committee established criteria for the prioritization and
selection of a SR topic (10). We developed and registered
a detailed protocol before undertaking the review, which
improved transparency of the review process. We searched
four foremost electronic databases, which cover most of
the literature in medicine and public health, why it is
unlikely that we may have missed any relevant literature.
Moreover, the review processes were thoroughly imple-
mented, with independent assessments taken at each stage
of the process, including literature screening and data
extraction.
One-third of the RCTs was graded as having a high risk
of bias, especially due to deviations from the intended
intervention, another third was graded having some con-
cerns regarding risk of bias, mainly arising from the ran-
domization process. Additional limitations include the
habitual diets in the RCTs, which may have affected the
ability to detect effects of the intervention. Moreover, the
animal protein in the RCTs was milk protein or casein,
which may not be totally representative for animal protein
sources. Among the RCTs, eight investigated soy protein
(27–31, 33–36) and ve other plant proteins, including
other legumes (25, 26, 29, 37) and grains (32). Although
overall results were not different for the different sources
of plant protein, it could be worth in future studies to
focus on other legumes and grains instead of soy. We did
not nd RCTs comparing other sources of plant protein
intake, than those above mentioned, to animal protein
intake in our search.
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Animal versus plant-based protein and risk of CVD and T2D
All included cohort studies were graded as having
a moderate risk of bias, which may constitute a lim-
itation of the underlying evidence. We extracted stud-
ies that reported on plant protein intake in relation to
animal protein intake, but this may, however, not cover
all possible sources of plant protein. Most of the stud-
ies were prone to limitations inherent in many obser-
vational epidemiologic studies – the starting time of
the exposure, method of assessment of dietary intake
as it was based on self-reported data (which, in addi-
tion, is usually done once at baseline), and inadequate
adjustment for confounding factors during the long fol-
low-up, thereby given a possibility for residual/unmea-
sured confounding across the reported estimates in the
studies.
Comparison with other SRs
We retrieved three previous SRs and meta-analyses
related to the comparison of animal protein intake to
plant protein intake or other diets with blood lipids as
outcomes in RCTs settings (45–47). Guasch-Ferré et al.
(45) included 36 RCTs, comparing diets with red meat to
diets that replaced red meat with a variety of foods. They
concluded that substituting red meat with high-quality
plant protein sources, but not with sh or low-quality
carbohydrates, leads to more favorable changes in blood
lipids and lipoproteins. Li et al. (46) included 104 RCTs,
also including individuals with, e.g., T2D and renal dis-
ease, comparing the effect of plant protein in substitu-
tion for animal protein on blood lipids. They concluded
that substitution of plant protein for animal protein
decreases LDL-cholesterol and non-HDL cholesterol.
Zhao et al. (47) focused on effects of plant protein and
animal protein on lipid prole as well as body weight
and body mass index in patients with conrmed hyper-
cholesterolemia. They concluded that compared with
animal protein, the consumption of plant protein could
improve lipid prole in patients with hypercholester-
olemia. Our results support the results from previous
SRs, even though we only included soy intake with low
concentrations of isoavones and subjects with normal
serum cholesterol concentrations or mild hypercho-
lesterolemia, which was reected in the low number of
studies included.
We found two previous SRs focused on protein intake,
including plant protein intake and risk of CVD mortality
(6, 7). Naghshi et al. (6) concluded that higher intake of
plant protein was associated with a lower risk of CVD
mortality, whereas there was no association of total pro-
tein or animal protein with the risk of CVD mortality. Qi
et al. reported (7) that higher plant protein intake (but not
total protein) was associated with a reduced risk of CVD
related- and all-cause mortality. In conclusion, our results
seem to be in line with these two SRs.
A previous SR and meta-analysis showed that total
protein and animal protein intake was associated with a
higher risk of T2D in both males and females, and that
plant protein decreased the risk of T2D in females. These
associations were also dependent on the food source, as
e.g. red meat and processed meat were risk factors of
T2D, while soy, dairy, and dairy products were protective
against T2D (48). Our results point in the same direction,
but we included fewer cohort studies, as the exposure
was dened as substitution of animal protein with plant
protein.
Altogether we found six recent SRs that could be con-
sidered comparable to the current paper (5, 6, 45–48).
The inclusion criteria were overall not exactly the same
as ours as we did not include interventions with soy con-
taining high or medium levels of isoavones, in contrast
to the previous reviews. In addition, we included only
prospective cohorts, which compared substitution of ani-
mal protein with plant protein, i.e. substitution analyses.
These differences in inclusion criteria lead to a lower num-
ber of included studies in comparison to previous SRs.
Interpretation and implications of ndings
The intervention studies showed signicantly, albeit only
small lowering of total cholesterol and LDL-cholesterol
along with higher HDL-cholesterol as a result of plant
protein intake in comparison with animal protein intake.
Soy, which has been studied extensively, may have a favor-
able effect on blood lipids, since it contains or can be for-
tied with high amount of isoavones, which are known
to have these effects (16). Although the magnitudes of
the differences in cholesterol levels were small, they may
be relevant in a life-course population perspective. The
results of the cohort studies indicated an association
between substitution of animal protein with plant pro-
tein on the risk of CVD and T2D. In comparison with
most animal protein sources, plant protein sources con-
tain less saturated fat and no cholesterol and more mono-
unsaturated and polyunsaturated fat, ber, antioxidants,
polyphenols, and other bioactive compounds (49). Other
mechanisms have also been suggested, i.e., related to
amino acid metabolism. Lysine, which is more prevalent
in animal proteins, has been shown to increase cholesterol
levels in animal models, whereas arginine, which is found
more in plant proteins, has been found to have the oppo-
site effect (47).
Conclusion
We found limited-suggestive evidence that substitution of
animal protein with plant protein may decrease the risk of
CVD mortality and T2D incidence. Protective effects seen
in RCTs on established risk factors for CVD supported
the evidence from observational studies. Replacement of
animal protein with plant protein for sustainability may
Citation: Food & Nutrition Research 2023, 67: 9003 - http://dx.doi.org/10.29219/fnr.v67.9003
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Christel Lamberg-Allardt et al.
also be considered as a public health strategy to lower
therisk of CVD and T2D.
Acknowledgments
The authors would like to thank university librarians
Sabina Gillsund and Gun-Brit Knutsön at Karolinska
Institutet for their invaluable assistance with the literature
searches, and the university librarians at the University of
Oslo for peer reviewing the search strategy.
Conict of interest and funding
Funding was received from the Nordic Council of
Ministers and governmental food and health authorities
of Norway, Finland, Sweden, Denmark, and Iceland.
Theauthors declare no conicts of interest.
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