261

Soy and the exercise-induced inflammatory

response in postmenopausal women
Kristen M. Beavers, Monica C. Serra, Daniel P. Beavers, Matthew B. Cooke, and
Darryn S. Willoughby

Abstract: Aging is associated with increasing inflammation and oxidative stress in the body, both of which can have nega-
tive health effects. Successful attenuation of such processes with dietary countermeasures has major public health implica-
tions. Soy foods, as a source of high-quality protein and isoflavones, may improve such indices, although the effects in
healthy postmenopausal women are not well delineated. A single-blind, randomized controlled trial was conducted in 31
postmenopausal women who were assigned to consume 3 servings of soy (n = 16) or dairy (n = 15) milk per day for
4 weeks. Parameters of systemic inflammation (tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), and interleukin-6
(IL-6)) and the oxidative defense system (superoxide dismutase (SOD), glutathione peroxidase, cyclooxygenase-2) were
measured post supplementation, before and after an eccentric exercise bout performed to elicit an inflammatory response.
A significant group-by-time effect for plasma TNF-a was observed (p = 0.02), with values in the dairy group increased
post supplementation and then decreasing into the postexercise period. Additionally, significant time effects were observed
for plasma SOD (p < 0.0001) and IL-6 (p < 0.0001) in the postexercise period. Overall results from our study do not sup-
port the notion that 4 weeks of daily soy milk ingestion can attenuate systemic elevations in markers of inflammation or
oxidative defense. However, data do suggest that the downhill-running protocol utilized in this study can be effective in al-
tering systemic markers of inflammation and oxidative defense enzyme activity, and that the ingestion of soy may help
prevent fluctuations in plasma TNF-a.
Key words: soy milk, inflammation, oxidative defense, downhill running, aging, isoflavone.
Résumé : Le vieillissement est associé à plus d’inflammation et de stress oxydatif dans l’organisme, ce qui entraine des effets
négatifs sur la santé. L’atténuation de ces effets au moyen de mesures alimentaires s’avère de la plus haute importance en
matière de santé publique. Les produits de soja, sources de protéines de grande qualité et d’isoflavones, recèlent un potentiel
d’amélioration de ces processus; cependant on ne connait pas bien les effets chez les femmes postménopausées en bonne
santé. Dans un essai clinique comparatif à simple insu et d’une durée de quatre semaines, on répartit au hasard 31 femmes
postménopausées; 16 acceptent de consommer tous les jours trois portions de produits de soja et 15, des produits laitiers. Les
paramètres de l’inflammation systémique (TNF-a, IL-1b, IL-6) et du système de défense contre l’oxydation (SOD, GPx,
COX-2) sont évalués après la supplémentation, et ce, avant et après une séance d’exercices pliométriques suscitant une
réponse inflammatoire. On observe un effet significatif du traitement en fonction du temps : les valeurs de TNF-a plasmatique
(p = 0,02) du groupe consommant des produits laitiers augmentent après la supplémentation puis diminuent après la séance
d’exercices. De plus, on observe un effet significatif en fonction du temps au sujet du SOD (p < 0,0001) et des IL-6 (p <
0,0001) plasmatiques après la séance d’exercices. D’après ces observations, la consommation quotidienne de boisson de soja
durant quatre semaines n’atténue pas les augmentations des marqueurs de l’inflammation et du système de défense antioxyda-
tive. En contrepartie, ces observations suggèrent que la course sur pente négative utilisée dans cette étude est efficace dans la
modification des marqueurs systémiques de l’inflammation et de l’activité enzymatique suscitée pour la défense antioxydative
et que la consommation de soja peut contribuer à la prévention des fluctuations plasmatiques de TNF-a.
Mots-clés : boisson de soja, inflammation, défense antioxydative, course en pente négative, vieillissement, isoflavone.
[Traduit par la Rédaction]

Received 21 September 2009. Accepted 18 February 2010. Published on the NRC Research Press Web site at apnm.nrc.ca on 13 May
2010.
K.M. Beavers,1,2 M.C. Serra,3 M.B. Cooke, and D.S. Willoughby. Department of Health, Human Performance, and Recreation, Baylor
University, Waco, TX. 76798-7313, USA.
D.P. Beavers.4 Department of Statistics, Baylor University, Waco, TX 76798-7313, USA.
1Corresponding author (e-mail: kbeavers@wfubmc.edu).
2Present address: Gerontology and Geriatric Medicine, Wake Forest University, School of Medicine, Medical Center Boulevard,
Winston-Salem, NC 27157, USA.
3Present address: Department of Gerontology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore,

MD 21201-1559, USA.
4Present address: Department of Biostatistical Sciences, Wake Forest University, School of Medicine, Medical Center Boulevard,

Winston-Salem, NC 27157-1063, USA.

Appl. Physiol. Nutr. Metab. 35: 261–269 (2010) doi:10.1139/H10-015 Published by NRC Research Press
262
Introduction
The process of senescence is associated with increasing
inflammation and oxidative stress in the body, both of which
play a role in the progression of cardiovascular disease
(CVD). Systemic inflammation is reflected, in part, by ele-
vations in proinflammatory cytokines. Elevations in these
cytokines, specifically tumor necrosis factor-a (TNF-a) and
interleukin-6 (IL-6), predict all-cause, as well as cardiovas-
cular, mortality (Volpato et al. 2001; Bradley 2008). Accu-
mulating evidence also indicates that oxidative stress plays
a major role in the initiation and progression of cardiovascu-
lar dysfunction (Dröge 2002; Taniyama and Griendling
2003), as the generation of reactive oxygen species (ROS)
causes endothelial cell apoptosis, increased monocyte adhe-
sion, and angiogenesis (Taniyama and Griendling 2003).
Sobering statistics suggest that CVD will affect more than
1 in 3 American adults during their lifetimes (Rosamond et
al. 2007). Although myocardial infarction risk is greater in
men, cerebrovascular accidents occur more commonly in
women, and in women over the age of 40 years, CVD is
the leading cause of death (Rosamond et al. 2007). This
risk especially increases after menopause and is thought to
be caused, at least in part, by decreased estrogen production.
Given the prevalence of CVD and the increased longevity of
women, CVD risk reduction in postmenopausal women rep-
resents a significant public health issue.
Identification of nutritional countermeasures that can at-
tenuate the progression of oxidative stress and inflammation
has major public health implications. Soy, as a unique
source of high-quality protein and isoflavones (phytoestro-
gens with antioxidant properties), is speculated to exert
such benefits, especially in postmenopausal women (Han et
al. 2002). Compelling in vitro and animal data support this
notion (Elia et al. 2006; Oh et al. 2007; Martinez-
Villaluenga et al. 2009), although data in humans are less
clear. Out of 6 studies assessing the effect of long-term (4
to 24 weeks) soy consumption on expression of IL-6 and
TNF-a (Jenkins et al. 2002; Hermansen et al. 2005; Hilpert
et al. 2005; Fanti et al. 2006; Ryan-Borchers et al. 2006;
Azadbakht et al. 2007), only 2 found a statistically signifi-
cant effect of soy, with 1 study finding an increase in the
expression of IL-6 (Jenkins et al. 2002) and the other a mod-
est decrease in TNF-a (Azadbakht et al. 2007).
Given the inconsistencies in the clinical literature,
coupled with the positive in vitro and animal data findings,
it has been suggested that basal concentrations of inflamma-
tory cytokines and the activities of oxidative stress–defense
biomarkers may be significant confounding factors in clini-
cal trial success. Dietary interventions may be efficacious
only in populations with elevations of such biomarkers at
baseline. Indeed, results from our laboratory suggest that
the mere consumption of soy milk for 4 weeks does not af-
fect the circulating concentrations of inflammatory cytokines
or activities of oxidative defense enzymes in a healthy pop-
ulation (Beavers et al. 2009). Therefore, studying these phe-
nomena in healthy individuals may require the employment
of methodology known to upregulate such processes in a
timely and consistent manner.
Intense physical exercise, especially that requiring eccen-
tric muscle contraction or new training regimens, causes in-
Appl. Physiol. Nutr. Metab. Vol. 35, 2010
flammation and muscular damage by increasing both muscle
proteases and ROS (Féasson et al. 2002; Peake et al. 2005b).
As little as 30 min of exercise elevates biomarkers of oxida-
tive stress in the plasma, thereby eliciting an acute-phase re-
sponse (Bloomer et al. 2005). With regard to soy
supplementation and acute exercise, the literature is scant
(Chen et al. 2004; Hill et al. 2004; Box et al. 2005),
although generally favorable. However, data on dietary in-
terventions using whole soy foods to achieve these outcomes
are lacking in the literature and, in light of increasing use, it
is important to understand the health effects of soy milk.
Therefore, the overall purpose of this study was to investi-
gate the effects of soy milk (compared with dairy milk) on
plasma markers of inflammation and oxidative defense in re-
sponse to a single bout of eccentric exercise designed to
elicit an acute-phase response in postmenopausal women.
Materials and methods
Study design
Table 1 shows the general research design and time
course of assessments administered in this study. The inde-
pendent variables were the supplement groups (soy milk or
dairy milk), the 4-week supplementation period, the eccen-
tric exercise protocol, and the blood-sampling times during
the course of this study. Dependent variables included the
information collected on the 24-h recall and 4-day food re-
cords, delayed-onset muscle soreness (DOMS), and plasma
inflammation and enzymatic oxidative defense variables
(TNF-a, interleukin-1b (IL-1b), IL-6, superoxide dismutase
(SOD), glutathione peroxidase (GPx), and cyclooxygenase-2
(COX-2)). Supplementation effects prior to eccentric exer-
cise have already been reported (Beavers et al. 2009).
Participants
An a priori power calculation showed that 14 participants
per group were necessary to detect a significant difference
between groups in markers of oxidative defense (SOD effect
size of 0.8 U
g–1; (Chen et al. 2004)) and inflammation
(IL-6 effect size of 0.24 pg
mL–1 (Jenkins et al. 2002)),
given a type I error rate of 0.05 and a power of 0.80. Partic-
ipants were recruited from the Central Texas area and were
considered eligible to participate in the study if they were
physically active but not trained (not engaged in an exercise
program involving either resistance or endurance
training >3 times per week for 1 year); were postmenopausal
(amenorrheic >1 year prior to start of study); and could ad-
here to the study protocol. Exclusionary criteria included ac-
tive use of hormone-replacement therapy, presence of
coronary artery diseases and (or) other significant uncon-
trolled cardiovascular, renal, hepatic, gastrointestinal, men-
tal, and endocrine disorders, including diabetes mellitus, a
body mass index (BMI) of <19 kg
m–2 or >35 kg
m–2, active
smoking status (within the past 3 years), an average intake
of >2 alcoholic drinks per day, and (or) the consumption of
any dietary supplements that could affect antioxidant status
(excluding multivitamins) 3 months prior to beginning the
study.
Familiarization session
Prior to starting the study, all participants went through a
Published by NRC Research Press
Beavers et al.
familiarization session to learn about the study protocol, ob-
tain medical clearance from their personal physician, and
sign university institutional review board-approved informed
consent documents. Participants were then assigned to begin
a 2-week washout period, in which they were instructed to
consume their usual diet but limit the consumption of all
soy-based or dairy-based products. Specifically, during the
study, each participant was instructed not to consume any
additional soy- or isoflavone-containing foods, to limit dairy
intake to 2 servings per day, and to avoid dairy milk. In ad-
dition, a 24-h recall was performed by a registered dietitian
utilizing a multiple pass method to determine ‘‘typical’’ cal-
orie and protein intakes (Rumpler et al. 2008).
Baseline assessment of maximum oxygen uptake and diet
Prior to beginning the supplementation protocol, each par-
ticipant was scheduled to come to the Exercise and Sport
Nutrition Laboratory at Baylor University (Waco, Tex.) for
baseline testing. During this session, participants had their
body mass, height, and BMI determined. Each subject was
then assessed for aerobic fitness by performing an incremental-
load exercise test of their maximal oxygen uptake
_ 2 max) on a treadmill ergometer (Quinton, Cardiac Sci-
(VO
ence, Bothell, Wash.). After baseline measurements at rest
were completed, the treadmill test was started at a velocity
of 8 km
h–1 and a 0% incline. The running speed was in-
creased by 2 km
h–1 every 3 min until exhaustion. Oxygen
uptake (VO_ 2) was measured every 30 s via an open-circuit
sampling system (Parvo Medics, Sandy, Utah). The test
was accepted as a VO _ 2 max if 2 of the following criteria
were met: (i) respiratory exchange ratio ‡ 1.15, (ii) rating
263
of perceived exertion (RPE) ‡ 19 on the RPE scale, and
(iii) maximum heart rate within 10 beats of age-predicted
maximum. The highest level of VO _ 2 was defined as
_ 2 max. Heart rate was calculated from a continuously
VO
monitored heart rate monitor (Polar Electro, Lake Success,
N.Y.) and blood pressure was determined with a mercurial
sphygmomanometer every 3 min during the exercise ses-
sion. Information from this test was used in participant
matching as well as in determining the speed to use during
the eccentric exercise bout.
Supplementation protocol
In a single-blind format, participants were matched for
_ 2 max) and baseline protein intake
physical fitness level (VO
–1 –1
(g
kg
day ), and randomly placed into 1 of 2 dietary
groups. Group A was instructed to consume a commercial
vanilla soy milk product and group B was instructed to con-
sume a commercial reduced-fat dairy milk product. The sup-
plement groups consumed 3 servings of their respective
supplements per day for a period of 4 weeks. Servings of
both supplements were matched as closely as possible for
total caloric (soy milk, 130 kcal (1 kcal = 4.186 kJ) vs. dairy
milk, 120 kcal) and protein content (soy milk, 6 g vs. dairy
milk, 8 g), with the soy milk providing an additional
*30 mg isoflavones per serving. Participants were asked to
record daily supplement consumption on an adherence log
provided to them at the baseline testing session. Finally, a
reported side-effects questionnaire was administered at 2, 4,
and 6 weeks to determine if any significant negative side ef-
fects occurred because of the dietary supplementation during
the course of the study.
Published by NRC Research Press
264
Dietary records
In an attempt to determine compliance with the dietary
prescriptions, and also to assess the average daily macronu-
trient consumption of fat, carbohydrate, and protein, each
participant was asked to keep 4-day dietary records during
weeks 2 and 6 of the study. Each participant was instructed
by a registered dietitian on how to fill out the record prior to
use. The dietary records were analyzed with the Food Pro-
cessor Dietary Assessment Software program (ESHA Re-
search Inc., Salem, Ore.).
Eccentrically biased downhill run
After the 4-week supplementation period, each participant
reported back to the laboratory for a downhill run–walk test.
Participants were instructed to refrain from aerobic exercise
for 48 h prior to the exercise session. A 5-min warm-up
(–10% grade and 3.5 km
h–1 speed) was performed on the
treadmill prior to the 45-min downhill running–walking ex-
ercise. The treadmill was then accelerated and participants
were instructed to run or walk at 60% of their VO_ 2 max (de-
termined from the baseline cardiopulmonary test) for
45 min. During the exercise protocol, 60% of VO _ 2 max was
maintained by measuring VO _ 2 every 3 min and adjusting
the treadmill speed accordingly. Between 15 and 18 min
and 27 and 30 min of exercise, heart rate and blood pressure
were recorded. After the exercise session, subjects donated
blood, received a small breakfast (granola bar (90 kcals and
1 g protein)), and remained rested until a 4-h blood sample
was taken.
Blood-collection procedure
Venous blood samples were drawn from the antecubital
vein into a 10-mL collection tube using a standard vacu-
tainer apparatus. Blood samples were allowed to stand at
room temperature for 10 min and were then centrifuged.
For each sample, the plasma was removed and frozen
at –80 8C for later analysis. Blood samples were collected
and analyzed after the 4-week supplementation period, im-
mediately post exercise, and at 4, 24, and 48 h after the ex-
ercise session. Except for the 4-h postexercise sample, all
blood samples were obtained after a 12-h fast and standar-
dized to the same time of day.
Assessment of plasma markers of inflammation and
oxidative stress
All biochemical analytical techniques were carried out ac-
cording to standard procedures (Orenstein et al. 2009; Row-
sey et al. 2009). Briefly, the activities of the plasma
oxidative defense enzymatic markers (SOD, GPx, and
COX-2) were assessed using enzyme-linked immunoabsorb-
ent assay (ELISA) technology with a microplate reader
(Wallac Victor-1420, Perkin-Elmer Life Sciences, Boston,
Mass.). TNF-a, IL-1b, and IL-6 concentrations were as-
sessed using the Bio-Plex bead-based multiplex assays
(Luminex xMAP technology, Bio-Rad Laboratories, Inc.,
Hercules, Calif.). All ELISA kit and Bio-Plex data were an-
alyzed in duplicate to reduce within-subject variability. The
intra-assay coefficients of variation for each biomarker were
as follows: SOD, 3.7%; COX-2, 8.7%; GPx, 7.2%; and
IL-1b, IL-6, TNF-a were all <20%. Data points falling
3 SDs above or below the group mean were labeled as out-
Appl. Physiol. Nutr. Metab. Vol. 35, 2010
Table 2. Baseline demographic statistics by treatment
group.
Soy group Dairy group
Variable (n = 16) (n = 15)
Age (y) 53.9±3.7 55.0±3.1
BMI (kg
m–2) 25.36±4.05 26.31±3.96
V̇O2 max (mL
kg–1min–1) 25.60±4.86 27.22±5.07
Protein intake (g
kg–1) 1.16±0.48 0.98±0.28
Note: Data are presented as means ± SD. No differences
were noted between treatment groups at baseline (all p > 0.05).
BMI, body mass index; V̇O2 max, maximal oxygen uptake.
liers (Stevens 2002) and those determined to be faulty by in-
strumentation error were excluded from analysis.
Statistical analyses
Baseline demographic, health, and dietary characteristics
were summarized as sample means and SDs. Means were
compared between groups using independent t tests to assess
the balance achieved via randomization. Study compliance,
defined as the percentage of prescribed beverage consumed,
was assessed across groups using a 2-sample Wilcoxon
rank-sum test because these data were demonstrably non-
normal. Statistical analysis for the biomarkers of interest
were performed by utilizing repeated-measures (group-by-
time) analyses of variance (ANOVAs) to control for intrain-
dividual variability. Because traditional post-hoc testing in a
repeated-measures framework does not fully account for in-
traindividual variability, significant differences in mean val-
ues for main effects or interactions were explored further
using profile plots and independent t tests. All statistical
procedures were performed using SAS, version 9.1.3 soft-
ware (Cary, N.C.) and a probability level of <0.05 was
adopted throughout.
Results
Retention and baseline characteristics
Thirty-three postmenopausal women were enrolled in this
study; one was unable to finish because of an unrelated
musculoskeletal injury and one was dropped because of diet-
ary noncompliance. Data from only those participants who
were at least 80% compliant with the dietary protocol (n =
31) were retained for later analysis. All baseline demo-
graphic data for these participants are presented in Table 2.
According to a Wilcoxon rank-sum test, there was no statis-
tically significant difference in dietary compliance between
the treatment groups (p = 0.54). Additionally, at baseline
there were no significant differences between the soy milk
and dairy milk group with regard to age (p = 0.37), BMI
(p = 0.52), cardiopulmonary fitness level (p = 0.37), or base-
line protein intake (p = 0.22). All study participants were of
Caucasian descent and no significant body weight changes
were observed over the course of the intervention (p =
0.22). All women participated in all blood draws (except for
1 participant who missed the immediately postexercise
blood draw) during the course of the study.
Reported side-effects questionnaires administered at base-
line and 2 and 4 weeks after consumption of soy or dairy
milk revealed minimal and similar side effects in the
Published by NRC Research Press
Beavers et al. 265

Table 3. Plasma oxidative stress and inflammatory marker data by treatment group.

Marker T1 T2 T3 T4 T5 p value
SOD
Soy 1.25±0.46 0.92±0.36 0.9±0.59 1.22±0.66 1.3±0.41 Group: 0.88
Dairy 1.09±0.38 1.05±0.44 0.9±0.57 1.33±0.47 1.29±0.48 Time: <0.0001*
GxT: 0.34
COX-2
Soy 1.74±1.23 1.76±1.65 1.74±1.57 1.83±1.60 2.07±1.82 Group: 0.18
Dairy 1.08±0.96 1.50±1.38 1.29±0.87 1.45±1.05 1.15±1.42 Time: 0.84
GxT: 0.62
GPx
Soy 140.24±32.31 141.57±25.24 143.4±28.80 148.44±35.30 151.71±19.22 Group: 0.24
Dairy 137.46±33.54 125.70±36.81 140.07±33.68 139.33±32.97 132.99±27.21 Time: 0.28
GxT: 0.42
TNF-a
Soy 2.71±1.01 2.77±1.16 2.86±1.02 3.24±1.52 2.75±0.72 Group: 0.31
Dairy 3.53±0.85 3.09±1.15 2.67±0.77 3.15±0.83 3.04±1.02 Time: 0.17
GxT: 0.04*
IL-1b
Soy 0.73±0.29 0.77±0.34 0.74±0.27 0.76±0.35 0.81±0.37 Group: 0.82
Dairy 0.77±0.22 0.75±0.22 0.78±0.22 0.7±0.23 0.73±0.30 Time: 0.81
GxT: 0.11
IL-6
Soy 2.10±0.78 2.95±1.43 4.68±2.78 2.69±1.50 2.39±1.26 Group: 0.71
Dairy 2.48±1.63 2.94±3.73 4.43±2.96 2.39±1.23 2.30±1.02 Time: <0.0001*
GxT: 0.43
Note: Data are presented as means ± SD. GxT denotes the group-by-time interaction, Group denotes the main effect for group, and
Time denotes the main effect for time. T1, pre-exercise; T2, immediately post exercise; T3, 4-h post exercise; T4, 24-h post exercise;
T5, 48-h post exercise; SOD; superoxide dismutase (U
mL–1); COX-2, cyclooxygenase-2 (pg
mL–1); GPx, glutathione peroxidase
(nmol
min–1
mL–1); TNF-a, tumor necrosis factor-a (pg
mL–1); IL-1b, interleukin-1b (pg
mL–1); IL-6, interleukin-6 (pg
mL–1).
*Significant (p < 0.05).

2 groups. All participants completed the required exercise Discussion

protocol and reported significant increases in DOMS (pre-
exercise, 0.69 ± 1.16 AU vs. 24 h post exercise, 4.17 ±
2.37 AU; p < 0.01). Lastly, no significant differences were
observed between groups for total daily caloric or macronu-
trient intake at baseline or post supplementation.
Effects of the intervention on select biomarkers
A 2-way (treatment groups (2)  time point (5)) repeated-
measures ANOVA to control for within-individual variation
was conducted to evaluate the effects of the different sup-
plements (soy milk or dairy milk) on the measured blood
markers. All plasma marker data are shown in Table 3, and
significant results are displayed pictorially in Fig. 1. Briefly,
no significant group effects were noted; however, a signifi-
cant group-by-time interaction was observed for plasma
TNF-a (p = 0.02) with values in the dairy group increased
post supplementation and then dropping through the post ex-
ercise period. TNF-a values for the soy group stayed more
consistent and slightly lower throughout most of interven-
tion period. Significant time effects (p < 0.0001) were ob-
served for plasma SOD and IL-6, with SOD activity
decreasing from pre-exercise (T1) to the 4-h (T3) time pe-
riod, and concentration of IL-6 increasing from pre-exercise
(T1) to the 4-h (T3) time period. No significant effects in
response to diet or exercise were seen for plasma GPx,
COX-2, or IL-1b.
Overall, our study does not support the notion that 4 weeks
of daily soy milk ingestion can attenuate systemic elevations
in markers of inflammation or oxidative defense in response
to eccentric exercise. However, data do suggest that the
downhill-running protocol utilized in this study can be effec-
tive in altering systemic activity and concentrations of select
biomarkers (specifically, SOD, TNF-a, and IL-6), and that
the ingestion of soy may help prevent fluctuations in plasma
TNF-a concentration after exposure to a stress-inducing
stimulus. Potential reasons for these findings are discussed
below and future research directions are presented
Plasma markers of oxidative stress
Harman (1956) proposed that ROS formed during normal
oxygen metabolism induce macromolecular damage and that
accumulation of such products accounts for the progressive
deleterious health changes we experience during aging. In
the body, the activity of certain antioxidant enzymes (e.g.,
SOD, GPx, and COX-2) can be assessed as indicators of the
oxidative stress imposed on the tissue.
In the present study, there was no significant effect of soy
supplementation on the activity of any of the plasma oxida-
tive defense enzymes assessed. However, we did note a time
effect for SOD, in which SOD activity was shown to de-
crease 4-h post exercise and return to baseline 2 days later.
This is contrary to what may be expected, because SOD ac-

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266 Appl. Physiol. Nutr. Metab. Vol. 35, 2010

Fig. 1. Column charts of significant changes in biomarker activity and concentration. Data are presented as means ± SD. From top, absolute
plasma superoxide dismutase (SOD) activity (U
mL–1), tumor necrosis factor-a (TNF-a) (pg
mL–1), and interleukin-6 (IL-6) (pg
mL–1) va-
lues are represented by time. T1, pre-exercise; T2, immediately post exercise; T3, 4-h post exercise; T4, 24-h post exercise; T5, 48-h post
exercise. The following sample sizes were observed for the soy group for T1 to T5, respectively: 16, 15, 15, 16, and 16. The following
sample sizes were observed for the dairy group for T1 to T5, respectively: 14, 15, 15, 15, and 15. *, Significant time effect (p < 0.0001);
{, significant group-by-time interaction (p < 0.05).

tivity is thought to increase during times of physical stress,
like downhill running (Hollander et al. 2001). However,
based on the increases in plasma IL-6 and the responses to
the DOMS questionnaires, it is believed that an inflamma-
tory state was induced, and that the decrease in the activity
of this marker may be due to a collective protective effect of
the supplements, or to inherent variability in measuring SOD
activity.
We could be observing a protective trend, with both the
soy and dairy milk products aiding in the conversion of
ROS to more inert byproducts, thereby decreasing the need
(and subsequent activity) of systemic SOD. From a dietary
standpoint, similarities do exist between the soy and dairy
milk products, including their carbohydrate, calcium, and vi-
tamin D composition, all of which may confer benefit. In a
recent animal study by Itoh et al. (2004), dietary calcium re-
striction was shown to significantly upregulate the activities
of manganese-SOD and copper zinc-SOD. Furthermore,
acute exercise, in addition to calcium restriction, decreased
both SOD isoenzymes in the diaphragms of calcium-
restricted rats. Thus, it is speculated that in our study, be-
cause of the calcium loading associated with consumption
of both beverages, SOD activity may have been suppressed,
which would explain the observed results. Unfortunately,
without a placebo group, we cannot infer the exact reason
for the time effect, although it is interesting to consider that
an unaccounted-for dietary factor may have played a role.
Furthermore, because the main decline in SOD activity
occurred 4-h post exercise, it may be that the carbohydrate-
rich granola bar given to both groups immediately after ex-

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Beavers et al.
ercise caused a blunting of SOD activity. There is limited
evidence in the animal literature to support this conjecture
(Lygren and Hemre 2002).
Additionally, the large variability in the activity of the en-
zymatic markers assessed in our study may have contributed
to this counterintuitive finding. Conflicting findings have
been reported with regard to SOD, with studies showing in-
creases (Buczynski et al. 1991; Chen et al. 1994), decreases
(Tozzi-Ciancarelli et al. 2002), and no change (Elosua et al.
2003) post exercise. Similar findings have also been reported
for GPx (Buczynski et al. 1991; Laaksonen et al. 1999;
Akova et al. 2001; Miyazaki et al. 2001; Vider et al. 2001).
In a comparably designed study, Chen et al. (2004) found
that isoflavone supplementation significantly increased pre-
exercise erythrocyte SOD activity and prevented the
exercise-induced decrease in activities of GPx. The authors
concluded that the results suggested that isoflavones can re-
store the redox homeostasis of antioxidant enzymes altered
because of exercise. Although the results from the present
study do not support the findings of Chen et al., differences
in the intervention product (isolated soy isoflavone vs.
whole soy food), isoflavone dosage (150 mg
d–1 vs.
*90 mg
d–1), and exercise modality (cycling vs. downhill
running) may explain the discrepancy.
Plasma markers of inflammation
The cytokines IL-1b, IL-6, and TNF-a are pleiotropic
molecules that play major roles in the inflammatory process
(Trikha et al. 2003; Bradley 2008) and are linked to cardio-
vascular morbidity (Mendall et al. 1997; Ridker et al.
2000a; 2000b) and mortality (Volpato et al. 2001). In our
study, we observed a significant group-by-time interaction
for the proinflammatory cytokine TNF-a, with postsupple-
mentation values of TNF-a elevated in the dairy group com-
pared with the soy group, followed by a decrease in group
mean values to T3 and a return to postsupplementation val-
ues for the dairy group by 24-h post exercise. The soy
group, however, did not fluctuate in the postsupplementation
or postexercise periods with regard to TNF-a concentration.
Thus it appears that with regard to this proinflammatory cy-
tokine, of which elevations are thought to promote chronic
disease, the soy group fared better overall than the dairy
group.
Although a significant group-by-time interaction was not
observed for plasma IL-6 values, a marked increase in post-
exercise IL-6 concentrations (specifically T3) was observed
in both groups, suggesting that the exercise bout success-
fully induced an acute inflammatory response. No time or
group trends were observed for plasma IL-1b; however, it
has been shown clearly that IL-6 rises in accordance with
both IL-1b and TNF-a, because each stimulates the synthe-
sis and release of IL-6 (Hildebrand et al. 2005). The synthe-
sis of IL-6 then dampens the synthesis of IL-1b and TNF-a
(Barton 1997). Therefore, it is speculative that there may
have been a time effect for IL-1b and TNF-a, although the
time points from which we sampled may not have corre-
sponded with the peak concentrations of these biomarkers.
Limitations
Although the present study contributes novel findings re-
garding the effects of soy consumption on the exercise-
267
induced inflammatory response in postmenopausal women,
there are limitations worthy of mention. Previous literature
does exist to support our chosen supplementation amount
and duration of study (Mitchell and Collins 1999; Djuric et
al. 2001), but success is clearly contingent on participant ad-
herence to the dietary protocol. Although self-report is an
accepted method for process evaluation (Jasti et al. 2005),
and adherence logs indicated excellent compliance among
both groups, because of funding constraints we do not have
any information regarding the isoflavone levels in biological
fluid (i.e., urine and blood). Moreover, the considerable
interindividual variability in the bioavailability of soy isofla-
vones (Hendrich 2002) may have contributed to the reported
lack of effect. Future studies should consider using more ob-
jective measures of adherence in their methodology, and
should account for individual differences in isoflavone bioa-
vailability when possible. Additionally, because SOD, GPx,
and COX-2 are enzyme components of the oxidative defense
system, and not direct markers of oxidative stress, it is
possible that we may not have captured the true effect of
soy supplementation on oxidative stress. Because of the var-
iability in assessing these enzymes, future studies may con-
sider assessing more direct measures (i.e., F2-isoprostanes or
8-oxo-2’-deoxyguanosine) of oxidative damage. Lastly,
although we selected sampling time points known to corre-
spond to peak concentrations–activities of biomarkers of in-
terest (Peake et al. 2005a; Peake et al. 2005b), it is possible
that we failed to capture the peak concentrations–activities
of all the biomarkers measured. Inferences, therefore, can
only be drawn from our sampling points.
Conclusions
Overall, results from our study do not support the notion
that 4 weeks of daily soy milk ingestion can attenuate sys-
temic elevations in markers of inflammation or oxidative de-
fense activity. However, data do suggest that the downhill-
running protocol utilized in this study can be effective in al-
tering systemic markers of inflammation and oxidative de-
fense. The findings from the plasma cytokine analysis
portion of our study add to a mixed body of literature that
hints at the potential of soy foods to influence proinflamma-
tory cytokines, although results are far from conclusive. Fu-
ture studies examining the ability of soy ingestion to reduce
systemic inflammation should address potential confounding
dietary factors by utilizing an inert control group to deter-
mine if a different nutritional compound found in both soy
and dairy milk could be a confounding, protective factor.
Despite the subdued findings in this study, the displacement
and lipid-lowering effects of soy consumption are well
known. Coupled with the potential for improvement in sys-
temic cytokine profiles, the results from this study still war-
rant inclusion of soy foods in a heart-healthy diet.
Acknowledgements
We thank Dr. Barbara J. Nicklas for thoughtfully review-
ing and significantly contributing to the completion of this
manuscript. Funding for this study was provided by White-
Wave Foods Inc., as well as by a dissertation grant provided
by Baylor University.
Published by NRC Research Press
268
References
Akova, B., Sürmen-Gür, E., Gür, H., Dirican, M., Sarandöl, E., and
Küçükoglu, S. 2001. Exercise-induced oxidative stress and mus-
cle performance in healthy women: role of vitamin E supple-
mentation and endogenous oestradiol. Eur. J. Appl. Physiol.
84(1–2): 141–147. doi:10.1007/s004210000331. PMID:
11394244.
Azadbakht, L., Kimiagar, M., Mehrabi, Y., Esmaillzadeh, A., Hu,
F.B., and Willett, W.C. 2007. Soy consumption, markers of in-
flammation, and endothelial function: a cross-over study in post-
menopausal women with the metabolic syndrome. Diabetes
Care, 30(4): 967–973. doi:10.2337/dc06-2126. PMID:17392557.
Barton, B.E. 1997. IL-6: insights into novel biological activities.
Clin. Immunol. Immunopathol. 85(1): 16–20. doi:10.1006/clin.
1997.4420. PMID:9325064.
Beavers, K.M., Serra, M.C., Beavers, D.P., Cooke, M.B., and
Willoughby, D.S. 2009. Soymilk supplementation does not alter
plasma markers of inflammation and oxidative stress in
postmenopausal women. Nutr. Res. 29(9): 616–622. doi:10.
1016/j.nutres.2009.09.002. PMID:19854376.
Bloomer, R.J., Goldfarb, A.H., Wideman, L., McKenzie, M.J., and
Consitt, L.A. 2005. Effects of acute aerobic and anaerobic exer-
cise on blood markers of oxidative stress. J. Strength Cond. Res.
19(2): 276–285. doi:10.1519/14823.1. PMID:15903362.
Box, W., Hill, S., and DiSilvestro, R.A. 2005. Soy intake plus
moderate weight resistance exercise: effects on serum concentra-
tions of lipid peroxides in young adult women. J. Sports Med.
Phys. Fitness, 45(4): 524–528. PMID:16446685.
Bradley, J.R. 2008. TNF-mediated inflammatory disease. J. Pathol.
214(2): 149–160. doi:10.1002/path.2287. PMID:18161752.
Buczynski, A., Kedziora, J., Tkaczewski, W., and Wachowicz, B.
1991. Effect of submaximal physical exercise on antioxidative
protection of human blood platelets. Int. J. Sports Med. 12(1):
52–54. doi:10.1055/s-2007-1024655. PMID:2030060.
Chen, C.Y., Bakhiet, R.M., Hart, V., and Holtzman, G. 2004. Iso-
flavones restore altered redox homesostasis of antioxidant en-
zymes in health young men undergoing 80% peak oxygen
consumption (VO2pk) exercise. Nutr. Res. 24(5): 347–359.
doi:10.1016/j.nutres.2004.02.001.
Chen, M.F., Hsu, H.C., and Lee, Y.T. 1994. Effects of acute exer-
cise on the changes of lipid profiles and peroxides, prostanoids,
and platelet activation in hypercholesterolemic patients before
and after treatment. Prostaglandins, 48(3): 157–174. doi:10.
1016/0090-6980(94)90016-7. PMID:7809382.
Djuric, Z., Chen, G., Doerge, D.R., Heilbrun, L.K., and Kucuk, O.
2001. Effect of soy isoflavone supplementation on markers of
oxidative stress in men and women. Cancer Lett. 172(1): 1–6.
doi:10.1016/S0304-3835(01)00627-9. PMID:11595123.
Dröge, W. 2002. Free radicals in the physiological control of cell
function. Physiol. Rev. 82(1): 47–95. PMID:11773609.
Elia, D., Stadler, K., Horváth, V., and Jakus, J. 2006. Effect of soy-
and whey protein-isolate supplemented diet on the redox para-
meters of trained mice. Eur. J. Nutr. 45(5): 259–266. doi:10.
1007/s00394-006-0593-z. PMID:16575496.
Elosua, R., Molina, L., Fito, M., Arquer, A., Sanchez-Quesada,
J.L., Covas, M.I., et al. 2003. Response of oxidative stress bio-
markers to a 16-week aerobic physical activity program, and to
acute physical activity, in healthy young men and women.
Atherosclerosis, 167(2): 327–334. doi:10.1016/S0021-9150(03)
00018-2. PMID:12818416.
Fanti, P., Asmis, R., Stephenson, T.J., Sawaya, B.P., and Franke,
A.A. 2006. Positive effect of dietary soy in ESRD patients with
systemic inflammation–correlation between blood levels of the
soy isoflavones and the acute-phase reactants. Nephrol. Dial.
Appl. Physiol. Nutr. Metab. Vol. 35, 2010
Transplant. 21(8): 2239–2246. doi:10.1093/ndt/gfl169. PMID:
16766544.
Féasson, L., Stockholm, D., Freyssenet, D., Richard, I., Duguez, S.,
Beckmann, J.S., and Denis, C. 2002. Molecular adaptations of
neuromuscular disease-associated proteins in response to ec-
centric exercise in human skeletal muscle. J. Physiol.
543(Pt. 1): 297–306. doi:10.1113/jphysiol.2002.018689. PMID:
12181300.
Han, K.K., Soares, J.M., Jr., Haidar, M.A., de Lima, G.R., and
Baracat, E.C. 2002. Benefits of soy isoflavone therapeutic regi-
men on menopausal symptoms. Obstet. Gynecol. 99(3): 389–
394. doi:10.1016/S0029-7844(01)01744-6. PMID:11864664.
Harman, D. 1956. Aging: a theory based on free radical and radia-
tion chemistry. J. Gerontol. 11(3): 298–300. PMID:13332224.
Hendrich, S. 2002. Bioavailability of isoflavones. J. Chromatogr. B
Analyt. Technol. Biomed. Life Sci. 777(1–2): 203–210. doi:10.
1016/S1570-0232(02)00347-1. PMID:12270213.
Hermansen, K., Hansen, B., Jacobsen, R., Clausen, P., Dalgaard,
M., Dinesen, B., et al. 2005. Effects of soy supplementation on
blood lipids and arterial function in hypercholesterolaemic sub-
jects. Eur. J. Clin. Nutr. 59(7): 843–850. doi:10.1038/sj.ejcn.
1602151. PMID:15900307.
Hildebrand, F., Pape, H.C., and Krettek, C. 2005. The importance
of cytokines in the posttraumatic inflammatory reaction. Unfall-
chirurg, 108(10): 793–794, 796–803. [In German.] doi:10.1007/
s00113-005-1005-1. PMID:16175346.
Hill, S., Box, W., and DiSilvestro, R.A. 2004. Moderate intensity
resistance exercise, plus or minus soy intake: effects on serum
lipid peroxides in young adult males. Int. J. Sport Nutr. Exerc.
Metab. 14(2): 125–132. PMID:15118187.
Hilpert, K.F., Kris-Etherton, P.M., and West, S.G. 2005. Lipid re-
sponse to a low-fat diet with or without soy is modified by C-
reactive protein status in moderately hypercholesterolemic
adults. J. Nutr. 135(5): 1075–1079. PMID:15867284.
Hollander, J., Fiebig, R., Gore, M., Ookawara, T., Ohno, H., and Ji,
L.L. 2001. Superoxide dismutase gene expression is activated by
a single bout of exercise in rat skeletal muscle. Pflugers Arch.
442(3): 426–434. doi:10.1007/s004240100539. PMID:11484775.
Itoh, M., Oh-Ishi, S., Hatao, H., Leeuwenburgh, C., Selman, C.,
Ohno, H., et al. 2004. Effects of dietary calcium restriction and
acute exercise on the antioxidant enzyme system and oxidative
stress in rat diaphragm. Am. J. Physiol. Regul. Integr. Comp.
Physiol. 287(1): R33–R38. PMID:14764436.
Jasti, S., Siega-Riz, A.M., Cogswell, M.E., Hartzema, A.G., and
Bentley, M.E. 2005. Pill count adherence to prenatal multivita-
min/mineral supplement use among low-income women. J.
Nutr. 135(5): 1093–1101. PMID:15867287.
Jenkins, D.J., Kendall, C.W., Connelly, P.W., Jackson, C.J., Parker,
T., Faulkner, D., and Vidgen, E. 2002. Effects of high- and low-
isoflavone (phytoestrogen) soy foods on inflammatory biomar-
kers and proinflammatory cytokines in middle-aged men and
women. Metabolism, 51(7): 919–924. doi:10.1053/meta.2002.
33352. PMID:12077742.
Laaksonen, D.E., Atalay, M., Niskanen, L., Uusitupa, M.,
Hänninen, O., and Sen, C.K. 1999. Blood glutathione homeosta-
sis as a determinant of resting and exercise-induced oxidative
stress in young men. Redox Rep. 4(1–2): 53–59. doi:10.1179/
135100099101534648. PMID:10714277.
Lygren, B., and Hemre, G. 2002. Influence of dietary carbohydrate
on antioxidant enzyme activities in liver of atlantic salmon.
Aquacult. Int. 9(5): 421–427. doi:10.1023/A:1020530432508.
Martinez-Villaluenga, C., Dia, V.P., Berhow, M., Bringe, N.A., and
Gonzalez de Mejia, E. 2009. Protein hydrolysates from beta-
conglycinin enriched soybean genotypes inhibit lipid accumula-
Published by NRC Research Press
Beavers et al.
tion and inflammation in vitro. Mol. Nutr. Food Res. 53(8):
1007–1018. doi:10.1002/mnfr.200800473. PMID:19603404.
Mendall, M.A., Patel, P., Asante, M., Ballam, L., Morris, J.,
Strachan, D.P., et al. 1997. Relation of serum cytokine concen-
trations to cardiovascular risk factors and coronary heart disease.
Heart, 78(3): 273–277. PMID:9391290.
Mitchell, J.H., and Collins, A.R. 1999. Effects of a soy milk sup-
plement on plasma cholesterol levels and oxidative DNA da-
mage in men–a pilot study. Eur. J. Nutr. 38(3): 143–148.
doi:10.1007/s003940050055. PMID:10443336.
Miyazaki, H., Oh-ishi, S., Ookawara, T., Kizaki, T., Toshinai, K.,
Ha, S., et al. 2001. Strenuous endurance training in humans re-
duces oxidative stress following exhausting exercise. Eur. J.
Appl. Physiol. 84(1-2): 1–6. doi:10.1007/s004210000342.
PMID:11394236.
Oh, H.Y., Lim, S., Lee, J.M., Kim, D.Y., Ann, E.S., and Yoon, S.
2007. A combination of soy isoflavone supplementation and
exercise improves lipid profiles and protects antioxidant de-
fense-systems against exercise-induced oxidative stress in ovar-
iectomized rats. Biofactors, 29(4): 175–185. doi:10.1002/biof.
5520290402. PMID:18057549.
Orenstein, S.B., Qiao, Y., Kaur, M., Klueh, U., Kreutzer, D.L., and
Novitsky, Y.W. 2009. Human monocyte activation by biologic
and biodegradable meshes in vitro. [Epub ahead of print.] Surg.
Endosc. 24(4): 805–811. doi:10.1007/s00464-009-0664-3.
Peake, J., Nosaka, K., and Suzuki, K. 2005a. Characterization of
inflammatory responses to eccentric exercise in humans. Exerc.
Immunol. Rev. 11: 64–85. PMID:16385845.
Peake, J.M., Suzuki, K., Wilson, G., Hordern, M., Nosaka, K.,
Mackinnon, L., and Coombes, J.S. 2005b. Exercise-induced
muscle damage, plasma cytokines, and markers of neutrophil ac-
tivation. Med. Sci. Sports Exerc. 37(5): 737–745. doi:10.1249/
01.MSS.0000161804.05399.3B. PMID:15870626.
Ridker, P.M., Hennekens, C.H., Buring, J.E., and Rifai, N. 2000a.
C-reactive protein and other markers of inflammation in the pre-
diction of cardiovascular disease in women. N. Engl. J. Med.
342(12): 836–843. doi:10.1056/NEJM200003233421202. PMID:
10733371.
Ridker, P.M., Rifai, N., Stampfer, M.J., and Hennekens, C.H.
2000b. Plasma concentration of interleukin-6 and the risk of fu-
ture myocardial infarction among apparently healthy men. Cir-
culation, 101(15): 1767–1772. PMID:10769275.
Rosamond, W., Flegal, K., Friday, G., Furie, K., Go, A.,
269
Greenlund, K., et al.; American Heart Association Statistics
Committee and Stroke Statistics Subcommittee. 2007. Heart dis-
ease and stroke statistics–2007 update: a report from the Ameri-
can Heart Association Statistics Committee and Stroke Statistics
Subcommittee. Circulation, 115(5): e69–e171. doi:10.1161/
CIRCULATIONAHA.106.179918. PMID:17194875.
Rowsey, P.J., Metzger, B.L., Carlson, J., and Gordon, C.J. 2009.
Long-term exercise training selectively alters serum cytokines
involved in fever. Biol. Res. Nurs. 10(4): 374–380. doi:10.1177/
1099800408329409. PMID:19190031.
Rumpler, W.V., Kramer, M., Rhodes, D.G., Moshfegh, A.J., and
Paul, D.R. 2008. Identifying sources of reporting error using
measured food intake. Eur. J. Clin. Nutr. 62(4): 544–552.
doi:10.1038/sj.ejcn.1602742. PMID:17426745.
Ryan-Borchers, T.A., Park, J.S., Chew, B.P., McGuire, M.K.,
Fournier, L.R., and Beerman, K.A. 2006. Soy isoflavones modu-
late immune function in healthy postmenopausal women. Am. J.
Clin. Nutr. 83(5): 1118–1125. PMID:16685055.
Stevens, J. 2002. Applied multivariate statistics for the social
sciences. Lawrence Erlbaum Associates, Mahwah, N.J.
Taniyama, Y., and Griendling, K.K. 2003. Reactive oxygen species
in the vasculature: molecular and cellular mechanisms. Hyper-
tension, 42(6): 1075–1081. doi:10.1161/01.HYP.0000100443.
09293.4F. PMID:14581295.
Tozzi-Ciancarelli, M.G., Penco, M., and Di Massimo, C. 2002. In-
fluence of acute exercise on human platelet responsiveness: pos-
sible involvement of exercise-induced oxidative stress. Eur. J.
Appl. Physiol. 86(3): 266–272. doi:10.1007/s00421-001-0542-8.
PMID:11990737.
Trikha, M., Corringham, R., Klein, B., and Rossi, J.F. 2003. Tar-
geted anti-interleukin-6 monoclonal antibody therapy for cancer:
a review of the rationale and clinical evidence. Clin. Cancer
Res. 9(13): 4653–4665. PMID:14581334.
Vider, J., Lehtmaa, J., Kullisaar, T., Vihalemm, T., Zilmer, K.,
Kairane, C., et al. 2001. Acute immune response in respect to
exercise-induced oxidative stress. Pathophysiology, 7(4): 263–
270. doi:10.1016/S0928-4680(00)00057-2. PMID:11228396.
Volpato, S., Guralnik, J.M., Ferrucci, L., Balfour, J., Chaves, P.,
Fried, L.P., and Harris, T.B. 2001. Cardiovascular disease, inter-
leukin-6, and risk of mortality in older women: the women’s
health and aging study. Circulation, 103(7): 947–953. PMID:
11181468.
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