1 ballard 2017 Effects of prior aerobic exercise on sitting-induced vascular dysfunction in healthy men

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Eur J Appl Physiol (2017) 117:2509–2518 
DOI 10.1007/s00421-017-3738-2
ORIGINAL ARTICLE
Effects of prior aerobic exercise on sitting-induced vascular 
dysfunction in healthy men
Kevin D. Ballard1  · Robert M. Duguid1 · Craig W. Berry1 · Priyankar Dey2 · 
Richard S. Bruno2 · Rose Marie Ward1 · Kyle L. Timmerman1 
Received: 11 July 2017 / Accepted: 5 October 2017 / Published online: 10 October 2017 
© Springer-Verlag GmbH Germany 2017
shear rate decreased at 3 h. In contrast, when sitting was 
preceded by EX, femoral artery FMD (2.7 ± 2.0%) and rest-
ing shear rate responses were unaffected. No between trial 
differences were detected for plasma glucose, MDA, ET-1, 
ARG, HA, ADMA, or SDMA.
Conclusion Prior aerobic exercise prevented the decline in 
femoral artery FMD that is otherwise induced by prolonged 
sitting independent of changes in oxidative stress, ET-1, and 
NO status.
Keywords Endothelin-1 · Flow-mediation dilation · 
Nitric oxide · Oxidative stress
Abbreviations
ADMA Asymmetric dimethylarginine
ANCOVA Analysis of covariance
ANOVA Analysis of variance
ARG Arginine
AUCSR Shear rate area under the curve
BMI Body mass index
DBP Diastolic blood pressure
ELISA Enzyme-linked immunosorbent assay
ET-1 Endothelin-1
EX Exercise
FMD Flow-mediated dilation
HA Homoarginine
HPLC High-performance liquid chromatography
MAP Mean arterial pressure
MDA Malondialdehyde
NO Nitric oxide
REST Quiet rest
SBP Systolic blood pressure
SDMA Symmetric dimethylarginine
TBA Thiobarbituric acid
VO2max Maximal oxygen consumption
Abstract 
Introduction Acute aerobic exercise prevents sitting-
induced impairment of flow-mediated dilation (FMD). Fur-
ther, evidence suggests that sitting-induced impairment of 
FMD occurs via an oxidative stress-dependent mechanism 
that disrupts endothelial function.
Purpose We hypothesized that acute aerobic exercise 
would prevent impairment of femoral artery FMD by lim-
iting oxidative stress responses that increase endothelin-1 
(ET-1) levels and disrupt nitric oxide (NO) status.
Methods In a randomized, cross-over study, healthy men 
(n = 11; 21.2 ± 1.9 years) completed two 3 h sitting trials 
that were preceded by 45 min of either quiet rest (REST) or 
a single bout of continuous treadmill exercise (65% maxi-
mal oxygen consumption) (EX). Superficial femoral artery 
FMD, plasma glucose, malondialdehyde (MDA), ET-1, 
arginine (ARG) and its related metabolites [homoarginine 
(HA), asymmetric dimethylarginine (ADMA), symmetric 
dimethylarginine (SDMA)] were assessed at baseline, 1 h 
following EX (or REST) (0 h), and at 1 h intervals during 3 h 
of uninterrupted sitting. Data were analyzed using repeated 
measures ANOVA.
Results During REST, femoral artery FMD declined from 
baseline (2.6 ± 1.8%) at 1, 2, and 3 h of sitting and resting 
Communicated by Mark Olfert.
 * Kevin D. Ballard 
 ballarkd@miamioh.edu
1 Department of Kinesiology and Health, College 
of Education, Health and Society, Miami University, 420 
South Oak Street, Oxford, OH 45056, USA
2 Human Nutrition Program, College of Education and Human 
Ecology, The Ohio State University, Columbus, OH 43210, 
USA
http://orcid.org/0000-0001-7587-1220
http://crossmark.crossref.org/dialog/?doi=10.1007/s00421-017-3738-2&domain=pdf
2510 Eur J Appl Physiol (2017) 117:2509–2518
1 3
Introduction
Sedentary behaviors, particularly time spent sitting, are 
common, with Americans spending 7.7 h/day in sedentary 
activity (Matthews et al. 2008). This is concerning as epi-
demiological evidence suggests a relationship between the 
amount of time spent sitting and cardiovascular disease 
(CVD) and all-cause mortality (Bauman et al. 2013; Chau 
et al. 2013; Ekelund et al. 2016; Patel et al. 2010). However, 
limited studies have provided a mechanistic understanding 
of the adverse effects of extended sitting on vascular health. 
Flow-mediated dilation (FMD) of the leg vasculature has 
emerged as a non-invasive technique to define the detri-
mental effects of prolonged sitting on endothelial function. 
Indeed, accumulating evidence in healthy adults suggests 
sitting-induced impairments in lower-body FMD are likely 
mediated by decreases in arterial shear stress (Morishima 
et al. 2016, 2017; Restaino et al. 2015, 2016; Thosar et al. 
2015a; Vranish et al. 2017). This suggests that lifestyle strat-
egies that increase shear stress may protect against sitting-
induced endothelial dysfunction.
Habitual aerobic exercise exerts numerous beneficial 
effects on the vasculature (Green et al. 2011). A single bout 
of aerobic exercise transiently increases arterial shear stress 
(Padilla et al. 2008a) and nitric oxide (NO) bioavailabil-
ity (Goto et al. 2007), likely contributing to observations 
of acute exercise-induced increases in upper- and lower-
body FMD (Currie et al. 2012; Harris et al. 2008; Totosy 
de Zepetnek et al. 2015; Tyldum et al. 2009). A recent study 
showed that 45 min of cycling transiently increased popliteal 
artery shear stress and prevented the decrease in FMD other-
wise induced by 3 h of sitting (Morishima et al. 2017). In the 
present study, we examined the efficacy of acute treadmill 
exercise to prevent reductions in superficial femoral artery 
shear stress and FMD responses to 3 h of sitting.
Further, indirect evidence in humans suggests that 
decreases in shear stress and consequent impairment of 
femoral artery FMD with 3 h of sitting occurs via an oxida-
tive stress-dependent mechanism (Thosar et al. 2015b) that 
disrupts NO bioavailability. Superficial femoral artery FMD 
reflects NO-dependent dilation (Kooijman et al. 2008). In 
contrast to the vasoprotective activities of NO, endothelin-1 
(ET-1) is a vasoconstrictor which contributes to the reduc-
tion in NO status associated with endothelial dysfunction 
(Iglarz and Clozel 2007) and is positively correlated with 
markers of oxidative stress (du Plooy et al. 2017). A better 
understanding of the mechanisms underlying lower-extrem-
ity endothelial dysfunction induced by sitting will advance 
our understanding of factors contributing to the preferen-
tial development of atherosclerosis in the leg vasculature 
(Kroger et al. 1999). Therefore, we additionally sought to 
determine the influence of 3 h of sitting on systemic oxida-
tive stress and biomarkers related to endothelial function 
(i.e., NO, ET-1) and if aerobic exercise preceding prolonged 
sitting exerts beneficial effects on these biomarkers. We 
hypothesized that a single bout of moderate-to-vigorous 
intensity treadmill exercise would prevent impairments in 
femoral artery endothelial function by improving plasma 
biomarkers of NO status that are otherwise disrupted by 
sitting.
Methods
Study design
The protocol for this study was approved by the Institutional 
Review Board at Miami University and written informed 
consent was obtained from all participants before enrolling. 
Healthy men (n = 11) completed a two-arm, randomized, 
cross-over study with trials separated by 1-week. Partici-
pants were recreationally active (≤ 3 days/week of moderate-
intensity physical activity) and enrolled on the basis of age 
(18–30 years), non-smoking status, and body mass index 
(BMI)brachial artery blood 
pressure were recorded two times using an automated blood 
pressure monitor (Omron HEM907XL, Bonnockburn, IL) 
following 5 min of seated rest. Mean arterial pressure (MAP) 
was calculated. Participants then performed a graded exer-
cise test on a motor driven treadmill to determine VO2max. 
Expired air was collected and analyzed using a calibrated 
Parvomedics True One 2400 Metabolic System (ParvoMed-
ics, Sandy, UT).
Approximately 1 week following the screening visit, 
participants reported to our laboratory in the fasted state 
(baseline) for superficial femoral artery FMD and MAP 
assessment and blood collection. Immediately thereafter, 
they performed 45 min of moderate-to-vigorous intensity 
(Garber et al. 2011) treadmill exercise (EX) or 45 min of 
quiet seated rest (REST) as a control condition. Femoral 
artery FMD, MAP, and venous blood samples were obtained 
1 h after EX (or REST) (0 h) and then hourly during 3 h of 
2511Eur J Appl Physiol (2017) 117:2509–2518 
1 3
sitting (Fig. 1). The two trials occurred at the same time of 
day (≤ 1 h).
Dietary control
Participants were instructed to complete a 1 day food log 
prior to their first testing trial and instructed to replicate their 
dietary intake the day prior to their second testing trial. Food 
records were assessed for energy and nutrient intake using 
Food Processor Nutrition Analysis software (Version 11.2, 
ESHA Research, Salem, OR).
Testing trials
Participants were seated for 10 min in a comfortable chair 
with back support before the assessment of baseline MAP 
and superficial femoral artery FMD. Next, a fasting blood 
sample was obtained from an antecubital vein, after which 
participants were provided with a standardized snack [Nature 
Valley oats and honey granola bar; 190 kcals; 29/7/3 g (car-
bohydrate/fat/protein)]. Consumption of the standardized 
snack was intended to minimize excursions in glycemia that 
are otherwise known to adversely affect endothelial function 
(Jacome-Sosa et al. 2016). Following 30 min of quiet seated 
rest, participants completed a 45 min bout of treadmill exer-
cise at a speed and grade corresponding to 65% VO2max or 
45 min of quiet seated rest. The aerobic exercise bout used in 
the present study was slightly lower in intensity, but similar 
in duration to prior studies showing greater 3 h post-exercise 
arterial shear rate responses following high-intensity (75% 
VO2max) compared to moderate- and low-intensity (25–50% 
VO2max) exercise (Padilla et al. 2008b). Our lower exer-
cise intensity was chosen to minimize increases in oxida-
tive stress during and immediately following high-intensity 
exercise (Goto et al. 2007) that may disrupt NO status and 
blunt endothelial function. During the EX trial, expired air 
was collected and analyzed using the same Parvomedics 
Metabolic System at 5 min intervals during the first 15 min 
of exercise to confirm that participants were within ± 5% of 
their individual 65% VO2max (Harris et al. 2008). Tread-
mill speed and/or grade were adjusted to maintain the cor-
rect intensity. After the subject demonstrated a stable VO2 
within the desired range, the mouthpiece was removed for 
the remaining duration of the exercise session. During the 
REST trial, participants sat quietly in the laboratory. Thirty 
minutes following the completion of EX (or REST), a sec-
ond snack that was identical to that consumed at baseline 
was provided.
Fifty minutes following EX (or REST), participants were 
seated in preparation for the 0 h MAP and FMD measures. A 
blood sample was collected from an antecubital vein imme-
diately following FMD. Participants then commenced the 
3 h uninterrupted sitting bout. Participants remained seated 
for 3 h in a comfortable chair with back support and were 
instructed to keep their feet resting on the floor and legs 
perpendicular to the ground. Study personnel monitored par-
ticipants throughout the prolonged sitting bout to ensure that 
no lower extremity movements occurred. MAP and femoral 
artery FMD were assessed and blood collected at 1 h inter-
vals during the 3 h sitting bout. Participants were allowed to 
use their arms to perform light activity (e.g. reading, work-
ing on a computer) during non-testing periods.
Superficial femoral artery flow mediated dilation
Superficial femoral artery FMD was assessed by high-
frequency ultrasonographic imaging at each time point as 
described (Thosar et al. 2015a), with minor modifications. 
In brief, the rapid inflation cuff was placed on the right lower 
thigh approximately 7 cm proximal to the knee joint and 
the superficial femoral artery was imaged longitudinally 
7–10 cm below the inguinal crease using a 5- to 12-MHz 
Fig. 1 Timeline of study measures. Superficial femoral artery flow 
mediated dilation (FMD) was assessed and blood obtained after an 
overnight fast (baseline), 1 h after 45 min of aerobic exercise (EX) or 
rest (REST) (0 h), and at 1 h intervals for 3 h of prolonged sitting. All 
measurements were made while the subject was in the seated posi-
tion. Participants ingested a standardized snack before and 30  min 
following the completion of EX (or REST)
2512 Eur J Appl Physiol (2017) 117:2509–2518
1 3
multi-frequency linear array transducer connected to a high-
resolution ultrasound (uSmart 3300; Terason, Burlington, 
MA). End-diastolic femoral artery diameter and continuous 
Doppler velocity were assessed simultaneously for 1 min. 
The thigh cuff was then inflated to 250 mmHg for 5 min 
using a rapid cuff inflator (Hokanson E20, Bellevue, WA), 
and then rapidly released. Recordings of arterial diam-
eter and blood velocity were obtained 1 min prior to cuff 
deflation and for 3 min thereafter at ten frames per second 
(Camtasia Studio, TechSmith Corporation, Okemos, MI). 
Participants’ skin was marked and the distance from the 
inguinal crease to the proximal edge of the transducer was 
recorded to ensure identical transducer placement for sub-
sequent FMD measures. Offline analyses were performed 
using automated edge-detection software with end-diastolic 
gating (Medical Imaging Applications, Iowa City, IA). All 
vascular measurements and analyses were performed by the 
same trained technician who was unaware of the treatments. 
From end-diastolic synchronized diameter (mm) and veloc-
ity data (m s− 1), shear rate was calculated as 4(velocity)/
diameter (Parker et al. 2009). Shear rate area under the curve 
 (AUCSR) was calculated from the time of cuff release until 
the time of maximal post-occlusion diameter to determine 
the hyperemic stimulus responsible for FMD (Pyke and 
Tschakovsky 2007). Consistent with prior findings (Tho-
sar et al. 2015a), dilation of the superficial femoral artery 
was not observed in at least one measurement time point in 
four participants during the EX trial and seven participants 
during the REST trial. Thus, the contribution of changes in 
 AUCSR to changes in FMD with sitting could not be defini-
tively established. The ultrasonographer for these experi-
ments (R. M. Duguid) has calculated reproducibility of 
superficial femoral artery FMD in our laboratory; the intra-
class correlation coefficient and coefficient of variation (CV) 
was 0.97 and 11.2%, respectively.
Materials
High-performance liquid chromatography (HPLC)-grade 
solvents and the following chemicals were purchased from 
Fisher Scientific (Pittsburgh, PA): methylmonoarginine, 
o-phthalaldehyde, and potassium phosphate. Thiobarbituric 
acid (TBA) was from Sigma (St. Louis, MO).
Blood processing and analyses
Blood was collected into evacuated tubes containing EDTA 
or lithium heparin. Whole blood was centrifuged (1500×g, 
4 °C, 15 min), plasma separated, and archived at − 80 °C. 
Plasma glucose was measured in accordance with the 
manufacturer’s instructions (Pointe Scientific, Canton, MI) 
on a microplate reader (BioTek Instruments, Synergy HT, 
Winooski, VT; intra-assay and inter-assay CV 3.1–5.0%). 
Plasma ET-1 was measuredusing an enzyme-linked immu-
nosorbent assay (ELISA) kit in accordance with the manu-
facturer’s instructions (R&D Systems, Minneapolis, MN; 
CV 4.1–5.5%).
Plasma malondialdehyde (MDA), a measure of lipid 
peroxidation, was measured by HPLC with fluorescence 
detection as described (Mah et al. 2011) following saponi-
fication, derivatization with TBA regeant, and subsequent 
extraction with butanol (CV 4.5–5.3%). Analysis was 
performed on a Shimadzu LC-20XR system (Shimadzu, 
Columbia, MD) equipped with a RF-10AXL fluorescence 
detector set to 532/553 nm (excitation/emission). Isocratic 
separation was performed at 0.8 mL/min on a Phenomenex 
Luna C18 column (250 × 4.6 mm i.d., 5 mm; Torrance, CA) 
using 50:50 methanol and 25 mmol/L phosphate buffer 
(pH 6.5) as the mobile phase. Plasma MDA concentrations 
were quantified from standards prepared in parallel from 
tetramethoxypropane.
Plasma arginine (ARG) (CV 3.0–5.1%) and its related 
metabolites [homoarginine (HA) (CV 2.3–4.3%), asym-
metric dimethylarginine (ADMA) (CV = 6.8–7.2%), and 
symmetric dimethylarginine (SDMA) (CV 7.4–8.0%)] were 
measured as indirect indicators of NO status as described 
(Mah et al. 2011). In brief, heparin plasma (100 μL) was 
subjected to solid phase extraction on a polymeric cation-
exchange column (Hypersep Retain-CX SPE column; 30 mg, 
1 mL; Fisher Scientific) using ammonia:water:methanol 
(10:40:50, v:v:v). Following evaporation under nitrogen 
gas, samples were reconstituted in water prior to incuba-
tion to generate O-phthalaldehyde-derivatives of arginine 
and ADMA. Separation was performed isocratically at 
1.3 mL/min using a Phenomenex Kinect XB-C18 column 
(50 × 3.0 mm i.d., 2.6 μm) on the aforementioned HPLC 
system programmed to 340/455 nm (excitation/emission) 
using 50 mmol/L potassium phosphate buffer (pH 6.5) and 
6.5% (v:v) acetonitrile as the mobile phase. Analytes were 
quantified on the basis of peak area relative to internal stand-
ard (methylmonoarginine) and the ratio of ADMA/ARG was 
calculated as an indirect index of NO status (Bode-Boger 
et al. 2007).
Statistical analyses
Sample size was determined utilizing data from a pre-
vious cross-over study showing that 3 h of sitting sig-
nificantly reduced superficial femoral artery FMD in 12 
young healthy men (Thosar et  al. 2015a). Data (Tho-
sar et al. 2015a) indicated that baseline femoral artery 
FMD responses did not differ between trials (4.6 ± 3.1%; 
mean ± SD) and decreased to 0.5 ± 0.9% at 1 h of pro-
longed sitting. Our power calculation indicated that a min-
imum of six participants would be needed in the present 
study in order to reject the null hypothesis with 80% power 
2513Eur J Appl Physiol (2017) 117:2509–2518 
1 3
(P 0.50; data not shown), indicat-
ing compliance to replicating their diets prior to each trial. 
Participants completed the 45 min of treadmill exercise at 
65.6 ± 3.9% VO2max (range 60.9–72.6%).
Femoral artery FMD
Baseline FMD responses did not differ between trials 
(Fig. 2). Significant main effects for time (PMDA, ARG, HA, ADMA, SDMA, and ADMA/
ARG did not differ between trials at baseline. A main effect 
for time (Ppopliteal artery shear rate and prevented the decrease in 
FMD otherwise induced by 3 h of sitting in healthy adults 
(Morishima et al. 2017). Our findings corroborate prior stud-
ies (Morishima et al. 2017), and extends them by showing 
that resting mean shear rate of the superficial femoral artery 
was higher 1 h following moderate-to-vigorous intensity 
treadmill exercise compared to the control trial. Thus, our 
data support that transient exercise-induced increases in 
arterial shear stress (Morishima et al. 2017; Padilla et al. 
2008a) contribute to the preservation of lower extremity 
FMD (Morishima et al. 2017) when acute exercise precedes 
prolonged sitting.
Limited studies have provided a mechanistic understand-
ing of the adverse effects of prolonged sitting on endothe-
lial function. Decreases in superficial femoral artery FMD 
induced by 3 h of uninterrupted sitting in 11 healthy men 
were prevented by ingestion of 1500 mg of the dietary anti-
oxidant vitamin C (Thosar et al. 2015b), suggesting that 
increased oxidative stress induced by low vascular shear 
stress (Malek et al. 1999) may contribute to the decline in 
lower extremity FMD during prolonged sitting (Morishima 
et al. 2016, 2017; Restaino et al. 2015, 2016; Thosar et al. 
2015a; Vranish et  al. 2017) by reducing NO bioavail-
ability. In our cohort of healthy men, we did not observe 
changes in systemic oxidative stress (i.e., MDA) during 3 h 
of uninterrupted sitting. Additionally, plasma levels of the 
vasoconstrictor ET-1 were unaffected by prolonged sitting, 
likely due to the lack of change in oxidative stress (du Plooy 
et al. 2017). Collectively, our data suggest that 3 h of unin-
terrupted sitting impairs superficial femoral artery FMD by 
an oxidative stress-independent mechanism. Further, plasma 
levels of ARG and its related metabolites were unaffected 
by sitting, suggesting possible preservation of NO status. In 
the present study, the ratio of ADMA/ARG was calculated 
as an indirect index of NO status. Future studies are needed 
in humans to directly validate ADMA/ARG as an indicator 
of NO status. To our knowledge, our study is the first to 
evaluate changes in circulating markers related to endothe-
lial function in response to prolonged sitting. Alternative 
mechanisms potentially explaining reductions in lower-body 
FMD with prolonged sitting have been suggested (Thosar 
et al. 2012), including increases in blood viscosity, inflam-
mation, and coagulation, and warrant investigation in future 
studies.
Our study was sufficiently powered to detect differences 
in superficial femoral artery FMD based on published data 
(Thosar et al. 2015a) and our sample size was similar to 
others examining lower extremity FMD (Restaino et al. 
2015, 2016; Thosar et al. 2015a). Only young men were 
tested in the present study, thus limiting the generalizability 
of our findings. A recent study found that popliteal artery 
FMD decreased in young men in response to 3 h of unin-
terrupted sitting, whereas FMD was unaffected in response 
to sitting in age-matched, pre-menopausal women (Vranish 
et al. 2017). Differences in FMD responses between sexes 
occurred despite comparable reductions in resting popliteal 
artery mean shear rate during 3 h of uninterrupted sitting 
(Vranish et al. 2017), suggesting selective impairment in 
microvascular function with prolonged sitting occurs inde-
pendent of sex. Future research is warranted to determine 
whether sitting-induced changes in endothelial function are 
influenced by age, training status, or chronic disease. In the 
present study, the standardized snack containing 29 g car-
bohydrates transiently increased plasma glucose by 12–15% 
at 0 h (i.e., 30 min following ingestion). We previously 
showed that ingestion of a beverage with a similar carbohy-
drate content transiently increased plasma glucose by 11% 
at 30 min postprandially. Brachial artery was unaffected by 
this limited hyperglycemic response (Ballard et al. 2013). 
Thus, we believe that ingestion of the second standardized 
snack (and limited resulting hyperglycemia) was unlikely to 
impact femoral artery FMD. Future studies are warranted 
to determine the combined influence of prolonged sitting 
and larger postprandial challenges on endothelial health. We 
did not observe changes in plasma levels of MDA, ET-1, or 
ARG and its metabolites, suggesting that systemic altera-
tions in oxidative stress, vasoconstriction, or NO status do 
not underlie decreases in superficial femoral artery FMD 
2517Eur J Appl Physiol (2017) 117:2509–2518 
1 3
with prolonged sitting. However, our data could also indicate 
that blood samples collected from the arm are not reflective 
of perturbations occurring in the lower body vasculature. 
Future studies should attempt to obtain blood samples from 
the lower body vessels that may better reflect local perturba-
tions in response to prolonged sitting.
This study advances existing knowledge that prolonged, 
uninterrupted sitting impairs lower-body endothelial func-
tion in healthy adults (Morishima et al. 2016, 2017; Restaino 
et al. 2015, 2016; Thosar et al. 2015a; Vranish et al. 2017) by 
showing that a prior bout of moderate-to-vigorous intensity 
aerobic exercise prevents impairments in superficial femoral 
artery FMD otherwise induced by 3 h of prolonged sitting. 
This is of public health importance, consistent with epidemi-
ological findings suggesting that increased daily sitting time 
is associated with CVD and all-cause mortality (Bauman 
et al. 2013; Chau et al. 2013; Ekelund et al. 2016; Patel et al. 
2010). Our data underscore aerobic exercise as an effective 
lifestyle strategy to attenuate sitting-induced leg endothelial 
dysfunction, a finding with practical implications for indi-
viduals whose occupations require long periods of sitting 
(e.g., long-distance travel, office workers). Prolonged sitting 
impaired femoral artery FMD without changing systemic 
markers related to endothelial function. Future studies are 
warranted to further explore the physiological mechanisms 
underlying sitting-induced endothelial dysfunction as well 
as evaluate various lifestyle factors (i.e., different exercise 
modalities, timing of exercise, dietary strategies) to attenu-
ate the deleterious effects of prolonged sitting on vascular 
health.
Acknowledgements This study was supported by a College of Edu-
cation, Health, and Society Seed Grant and Miami University Under-
graduate Summer Scholars Program. The authors acknowledge the 
contributions of our participants and Kristina Arslain for her assistance 
with diet record entry.
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	Effects of prior aerobic exercise on sitting-induced vascular dysfunction in healthy men
	Abstract 
	Introduction 
	Purpose 
	Methods 
	Results 
	Conclusion 
	Introduction
	Methods
	Study design
	Dietary control
	Testing trials
	Superficial femoral artery flow mediated dilation
	Materials
	Blood processing and analyses
	Statistical analyses
	Results
	Participants and dietary intakes
	Femoral artery FMD
	Plasma glucose and endothelin-1 (Table 3)
	Plasma oxidative stress and nitric oxide status (Table 3)
	Discussion
	Acknowledgements 
	References