Clinical and Experimental Pharmacology and Physiology (2003) 30, 382–386
EFFECTS OF MILD AEROBIC PHYSICAL EXERCISE ON
MEMBRANE FLUIDITY OF ERYTHROCYTES IN
ESSENTIAL HYPERTENSION
Kazushi Tsuda, Akiyoshi Yoshikawa, Keizo Kimura and Ichiro Nishio
Division of Cardiology, Department of Medicine, Wakayama Medical University, Wakayama, Japan
SUMMARY
1. The present study was undertaken to investigate the
effects of aerobic physical exercise on membrane function in
mild essential hypertension.
2. Hypertensive patients were divided into an exercise group
(n = 8) and a non-exercise (control) group (n = 8). Physical
exercise within the intensity of the anaerobic threshold level
was performed twice a week for 6 months. Membrane fluidity
of erythrocytes was examined by means of electron para-
magnetic resonance (EPR) and spin-labelling methods before
and after the trial period in both groups.
3. After physical exercise, blood pressure decreased signifi-
cantly.
4. Compared with the non-exercise group, in the exercise
group both the order parameter (S) and the peak height ratio
(ho/h-1) in the EPR spectra of erythrocytes were significantly
reduced (S, 0.717  0.004 vs 0.691  0.008, respectively
(n = 8), P < 0.05; ho/h-1, 5.38  0.06 vs 4.89  0.06, respectively
(n = 8), P < 0.05). These findings indicated that exercise
increased membrane fluidity and improved the membrane
microviscosity of erythrocytes.
5. There was no direct correlation between blood pressure
reduction and the exercise-induced increase in membrane
fluidity of erythrocytes.
6. In the non-exercise (control) group, blood pressure and
membrane fluidity were not changed after a 6 month follow-up
period.
7. The results show that aerobic physical exercise increased
erythrocyte membrane fluidity and improved the rigidity of
cell membranes in hypertensive patients. The improvement of
rheological properties of erythrocytes may explain, in part, the
cellular mechanisms for the beneficial effects of physical
exercise in hypertension.
Key words: aerobic physical exercise, electron paramagnetic
resonance, erythrocytes, essential hypertension, membrane
fluidity.
Correspondence: Dr Kazushi Tsuda, Division of Cardiology, Department
of Medicine, Wakayama Medical University, Kimiidera 811-1, Wakayama
641-8509, Japan. Email: tsudak@mail.wakayama-med.ac.jp
Received 30 July 2002; revision 12 November 2002; accepted 10 January
2003.
INTRODUCTION
Many studies have shown that regular physical exercise is bene-
ficial for both the prevention and treatment of hypertension and
that it can reduce cardiovascular disease morbidity and mortality.1,2
Cade et al.3 demonstrated that an exercise programme based on
walking for 3 months induced a significant reduction in blood
pressure in subjects with mild or moderate essential hypertension.
Van Hoof et al.4 reported that, using 24 h ambulatory blood
pressure monitoring, daytime blood pressure was significantly
decreased after a 4 month physical training period. Therefore,
physical exercise is recommended as a non-pharmacological treat-
ment for mild hypertension.5
It has been proposed that a cell membrane abnormality may be
an aetiological factor in hypertension.6–10 This includes not only
functional abnormalities, such as transmembrane cation fluxes,6,7
but also structural or physical changes of the cell membranes.8–10
Recently, an electron paramagnetic resonance (EPR) and spin
labelling method has been developed to elucidate membrane
fluidity and perturbations of membrane function by external
agents.11–13 Membrane fluidity is a physicochemical feature of
biomembranes and has an important role in modulating cell
functions, such as rheological behaviour and membrane micro-
viscosity.13,14 Using the EPR method, we showed that the mem-
brane fluidity of erythrocytes was lower in both spontaneously
hypertensive rats (SHR) and patients with essential hyper-
tension.15–17 However, the effects of physical exercise on mem-
brane function in hypertension are not yet clear.
In the present study, in order to determine the cellular mechan-
isms underlying the beneficial effects of aerobic physical exercise,
we investigated changes in the membrane fluidity of erythrocytes
before and after a trial period of exercise in essential hypertension
using the EPR method.
METHODS
Subjects
We studied 16 Japanese men with mild untreated essential hypertension
(systolic blood pressure (SBP) > 140 mmHg and/or diastolic blood pres-
sure (DBP) > 90 mmHg) who had exercise habits. There were no important
differences in physical activities among the subjects investigated. Patients
with a history of secondary hypertension, coronary heart disease, conges-
tive heart failure, lung diseases, cerebral complications, renal insufficiency,
diabetes mellitus, dyslipidaemia and haematological or hepatic disorders
were excluded from the study. Written consent was obtained from all
participants after the nature and objective of the study had been explained
to them.
 Physical exercise and membrane fluidity 383

Eight patients (mean (SEM) age 46.2  1.4 years) were subjected to
regular aerobic exercise. A 4 week run-in period was followed by a 6 month
physical exercise period. Eight patients (age 49.0  5.1 years) were sub-
jected to 6 months of follow up without any life-style modification. During
the run-in period, subjects remained sedentary and blood pressures were
stable. The patients were divided randomly into an exercise group and a
non-exercise (control) group. Before the trial period of exercise, we meas-
ured the fitness of the subjects and performed a physical examination, as
well as routine laboratory investigations of blood, electrocardiography,
chest and abdominal X-rays and echocardiography. There were no signifi-
cant differences in age, blood pressure and routine laboratory findings
between the exercise and the non-exercise groups (Table 1).
Exercise intensity
In order to determine the individual exercise intensity in the exercise group,
an initial treadmill exercise test (Stress Test System ML-5000 and
MAT-2000; Fukuda Denshi, Tokyo Japan) with respiratory gas exchange
monitoring (Metabosystem IS-5000; Fukuda Denshi) was performed before
the study and the anaerobic threshold (AT) level was obtained in each
subject according to the method. described by Wasserman et al.18 In the
preliminary study, we examined Vo2 values at both the AT and peak exercise
levels in hypertensive subjects (18.3  0.8 and 34.4  1.7 mL/min per kg,
respectively; n = 14). The workload at the heart rate of the initial AT level
was used as the exercise intensity in each subject.
Exercise protocols
The physical exercise consisted of a mild programme (10 min warm up,
15 min jogging at the heart rate of the AT level, 5 min walking and 30 min
calisthenics). The exercise was performed twice a week for 6 months in our
laboratory. The participants were supervised by the authors during the
exercise programme.
In the preliminary study, we ascertained the effects of the same intensity
of exercise on cardiac diastolic function in hypertensive subjects by using
echocardiography. The time of IIA-mitral valve opening on echocardio-
graphy (IIA-MVO) was significantly decreased after exercise (72.2  3.2
vs 59.9  4.2 msec; n = 13; P < 0.05), which may indicate that the exercise
was effective and improved cardiac diastolic function in hypertensive
subjects.
In both the exercise and non-exercise groups, the daytime blood pressure
at rest was measured indirectly with a cuff and a mercury sphygmomano-
meter in a sitting position before and after the trial period. The blood
pressure measurement was performed three times in each subject after
30 min rest while fasting and the mean of the values is reported.
Measurement of membrane fluidity of erythrocytes
Blood sampling was also performed before and after the trial period in all
subjects. Blood sampling was done by venipuncture after a minimum
30 min rest while fasting. After the plasma and buffy coat had been removed
carefully by centrifugation at 155 g for 10 min at 4C, washed erythrocytes
were resuspended in isotonic buffer (140 mmol/L NaCl, 20 mmol/L Tris-
HCl, pH 7.4) at a haematocrit of 50%. A 100 L aliquot of a solution
containing fatty acid spin label agents (5-nitroxide stearate (5-NS) and 16-
nitroxide stearate (16-NS); 5  10−5 mol/L) was added to the 200 L
erythrocyte suspension and the mixed solution was then incubated for 2 h
at 37C with gentle shaking.
The EPR measurements were performed using an EPR spectrometer
(model JEOL JES-FE2XG; Nihon Denshi, Tokyo, Japan) with a microwave
unit (model JEOL ES-SCXA; Nihon Denshi). The microwave power was
5 mW and the modulation frequency was 100 kHz, with a modulation
amplitude of 2.0 gauss (G). The temperature of the measurement was
controlled at 30C. The receiver scan width was 3280  50 G, with a sweep
time of 8 min, and the receiver gain was 4  103−7.9  103, with a
response time of 1.0 s. The fatty acid spin label agents are believed to be
anchored at the lipid–aqueous interface of the cell membranes by their
carboxyl ends, whereas the nitroxide group moves rapidly through a
restricted angle around the point of attachment.11,12,15–17 Therefore, the EPR
spectra of the fatty acid spin label agents are used to detect an alteration in
the freedom of motion in biological membranes and to provide an indic-
ation of membrane fluidity, which is inversely correlated with membrane
microviscosity. For indices of membrane fluidity, we have evaluated the
values of outer and inner hyperfine splitting (2T// and 2T⊥ in G, respec-
tively) in the EPR spectra for 5-NS and calculated the order parameter (S)
from 2T// and 2T⊥15–17 (Fig. 1). In the EPR spectra for 16-NS, we used
the peak height ratio (ho/h-1) value as an index of membrane fluidity15–17
(Fig. 1). The order parameters (S) may represent the fluidity of the outer
layer of the plasma membranes, whereas the peak height ratio (ho/h-1) may
express the fluidity of the inner membrane compartment.15 The intra-assay
coefficient of variation of the order parameter (S) and the peak height ratio
(ho/h-1) was less than 0.50%. In a preliminary study, we also examined the
interassay variation of the membrane fluidity of erythrocytes in hyper-
tensive subjects within 1 week. The alterative degree was −0.25 ± 0.23%
of basal value (n = 7). Lesser values of the order parameter (S) and peak
height ratio (ho/h-1) are associated with greater freedom of motion of the

Table 1 Characteristics of the exercise and non-exercise groups before and after the trial period of physical exercise

Exercise group (n = 8) Non-exercise group (n = 8)

Before After Before After

BMI (kg/m2) 25.2 ± 0.80 24.7 ± 0.6 24.9 ± 1.10 24.9 ± 1.00
Heart rate (b.p.m.) 69 ± 30 68 ± 2 72 ± 40 70 ± 30
SBP (mmHg) 144 ± 400 134 ± 2*† 148 ± 200 149 ± 300
MBP (mmHg) 111 ± 200 104 ± 1* 110 ± 300 109 ± 400
DBP (mmHg) 95 ± 20 89 ± 1* 91 ± 40 90 ± 50
Erythrocyte counts (104 cells/L) 520 ± 110 502 ± 12 500 ± 240 500 ± 220
Haemoglobin (g/dL) 15.6 ± 0.20 15.6 ± 0.3 15.0 ± 0.50 15.0 ± 0.40
Haematocrit (%) 48.0 ± 1.00 47.8 ± 1.0 45.0 ± 2.00 45.0 ± 2.00
Serum sodium (mmol/L) 142.3 ± 0.400 142.7 ± 0.40 141.7 ± 1.200 140.5 ± 0.500
Serum potassium (mmol/L) 4.2 ± 0.1 4.2 ± 0.1 4.0 ± 0.1 4.0 ± 0.1
Serum creatine (mg/dL) 1.0 ± 0.1 1.0 ± 0.1 0.8 ± 0.1 0.9 ± 0.1
Total cholesterol (mg/dL) 210 ± 110 210 ± 10 220 ± 160 208 ± 150
HDL-C (mg/dL) 51 ± 20 48 ± 1 49 ± 30 50 ± 30
Triglyceride (mg/dL) 110 ± 170 110 ± 11 130 ± 270 130 ± 280
Fasting serum glucose (mmol/L) 6.2 ± 0.2 6.1 ± 0.3 6.1 ± 0.4 5.9 ± 0.3
Serum ionized calcium (mmol/L) 1.26 ± 0.03 1.28 ± 0.01 1.21 ± 0.02 1.20 ± 0.03
Data are the mean±SEM. *P < 0.05 compared with before exercise; †P < 0.05 compared with the non-exercise group.
BMI, body mass index; SBP, MBP, DBP, systolic, mean and diastolic blood pressure, respectively; HDL-C, high-density lipoprotein–cholesterol.
384 K Tsuda et al.
spin labels in the biomembrane bilayers, indicating increased membrane
fluidity.15–17
Haematological and humoral parameters
Blood erythrocyte counts and other routine laboratory characteristics were
examined before and after the trial period. Plasma noradrenaline (HPLC
with an electrochemical detector) was also measured in the exercise group.
Fig. 1 Typical electron paramagnetic resonance (EPR) spectra of erythro-
cytes for the fatty acid spin-label agents (5-nitroxide stearate and 16-nitro-
xide stearate). S, order parameter; 2T// (in gaus (G)), outer hyperfine
splitting; 2T⊥ (in G), inner hyperfine splitting; Tzz and Txx, hyperfine con-
stants; an/an, isotropic coupling constant; ho/h-1, peak height ratio. Lesser
values of the order parameter (S) and the peak height ratio (ho/h-1) are
associated with greater freedom of motion of the spin labels in the bio-
membrane bilayers, indicating increased membrane fluidity.
Table 2 Membrane fluidity of erythrocytes (order parameters (S) and peak height ratio (ho/h1)) in hypertensive patients before and after the trial period of
exercise
Exercise group (n = 8)
Before
Order parameter (S) 0.717 ± 0.004 0.691 ± 0.008*†
Peak height ratio (ho/h1) 5.38 ± 0.06 4.89 ± 0.06*†
Data are the mean±SEM. *P < 0.05 compared with before exercise; †P < 0.05 compared with the non-exercise group.
Blood sampling for the measurement of these parameters was performed at
the same time as erythrocyte sampling for the EPR investigation.
Materials for EPR measurements
The spin label agents 5-NS and 16-NS were purchased from Aldrich
(Milwaukee, WI, USA). All other drugs were standard laboratory reagents
of analytical grade.
Statistical analysis
Values are expressed as the mean±SEM. Baseline parameters before and
after the exercise period were compared using one-way analysis of variance
(ANOVA) followed by Student’s paired t-test. Differences between the
different groups were analysed with Student’s unpaired t-test. P < 0.05 was
accepted as significant.
RESULTS
Effects of physical exercise on blood pressure and
humoral parameters in patients with mild essential
hypertension
After the 6 month exercise period, the daytime blood pressure at
rest decreased significantly in the exercise group (Table 1). In
particular, the reduction was pronounced for SBP (Table 1). The
blood pressure in the non-exercise group was not changed after
a 6 month follow-up period (Table 1). The body mass index tended
to decrease in the exercise group, although the reduction was not
significant (Table 1). In addition, there were no significant changes
by the exercise in blood erythrocyte counts, haemoglobin,
haematocrit, serum sodium and potassium, serum creatinine, serum
total cholesterol and high-density lipoprotein–cholesterol, serum
triglyceride and fasting plasma glucose (Table 1).
There were no significant changes in the serum free Ca level in
both the exercise and non-exercise groups (Table 1).
The plasma noradrenaline level decreased slightly in the exercise
group (273 ± 43 vs 218 ± 24 pg/mL; n = 8), although the change
was not significant.
Effects of physical exercise on membrane fluidity of
erythrocytes in essential hypertension
Table 2 gives the membrane fluidity of erythrocytes before and
after the trial period in the exercise and non-exercise groups. The
values of the order parameter (S) and the peak height ratio were
significantly reduced in the exercise group.
This finding indicated that physical exercise increased the
membrane fluidity of erythrocytes in hypertensive patients.
However, there was no direct correlation between the reduction
in blood pressure and the membrane fluidity of erythrocytes follow-
ing exercise. In addition, there was no significant correlation
Non-exercise group (n = 8)
After Before After
0.725 ± 0.004 0.726 ± 0.004
5.22 ± 0.04 5.23 ± 0.04
 Physical exercise and membrane fluidity
between the changes in plasma noradrenaline content and the
membrane fluidity of erythrocytes in the exercise group.
In the non-exercise group, the order parameter (S) and the peak
height ratio (ho/h-1) were not significantly changed after a 6 month
follow-up period (Table 2).
DISCUSSION
Many studies have suggested that regular exercise is beneficial for
the prevention and treatment of hypertension and that exercise can
lower both SBP and DBP in subjects with mild essential hyper-
tension.1–4 The American College of Sports Medicine proposes that
not only does adequate endurance exercise training consist of a
.
programme at an intensity of 50–85% of Vo2max, but also that less
intense exercise may similarly lower blood pressure.19 Physical
exercise may also attenuate other cardiovascular risk factors, such
as dyslipidaemia and insulin resistance.20–22 The present study also
demonstrated that mild aerobic physical exercise at the heart rate
of the AT level for 6 months significantly lowered blood pressure
in subjects with essential hypertension.
It is well known that membrane fluidity may be inversely
correlated with membrane microviscosity and rigidity.13 Erythro-
cyte membrane functions are critically important for rapid and
homogeneous perfusion of oxygen in the microcirculation.23
Previously, we have shown that erythrocyte membrane fluidity is
decreased in SHR and in patients with essential hypertension
compared with normotensive controls and we suggested that
cell membranes may be stiffer and less fluid in primary hyper-
tension.15–17 However, the effects of exercise on membrane function
are unclear. Therefore, in the present study, we examined changes
in membrane fluidity of erythrocytes before and after physical
exercise in mild hypertensive patients using the EPR method. The
order parameter (S) and the peak height ratio (ho/h-1) of the EPR
spectra obtained from erythrocyte membranes decreased signifi-
cantly after the trial period of physical exercise in hypertensive
patients. The result indicates that exercise increased membrane
fluidity and improved the membrane microviscosity of erythro-
cytes. To our knowledge, this is the first report showing that aerobic
physical exercise affects the membrane fluidity of erythrocytes in
hypertensive subjects.
The lowering of the microviscosity and, consequently, the
increased flexibility of the membranes may play an important role
in erythrocyte diffusion in the microcirculation. Therefore, it is
possible that physical exercise may improve the microcirculation
and could contribute to the beneficial effects for the prevention of
cardiovascular complications in hypertension. In the present study,
there was no direct correlation between the reduction in blood
pressure and changes in the membrane fluidity of erythrocytes
following exercise. McLaren et al.24 also reported that membrane
fluidity did not correlate with blood pressure in SHR. It seems
likely that the exercise-induced increase in the membrane fluidity
of erythrocytes may not be a consequence of the reduction in blood
pressure . There have been no reports regarding the effects of
exercise on the membrane fluidity of erythrocytes in healthy
normotensive subjects. Additional studies should be performed to
assess more thoroughly the relationship between blood pressure
changes and the membrane fluidity of erythrocytes.
The underlying mechanism of exercise-inducecd changes in
membrane fluidity remains uncertain. It is possible that exercise
385
may be associated with intravascular haemolysis leading to
anaemia. Although there were no significant differences in erythro-
cyte counts, haemoglobin and haematocrit before and after exercise
in the present study, the possibility of exercise-induced micro-
haemolysis in blood vessels cannot be fully excluded.
Recently, it has been shown that aerobic physical exercise may
reduce sympathetic nervous activity in hypertension.25 In the
present study, plasma noradrenaline was slightly decreased in the
exercise group, although the change was not statistically signifi-
cant. In addition, there was no significant correlation between
changes in plasma noradrenaline and the membrane fluidity of
erythrocytes in the exercise group.
Sumikawa et al.26 reported changes in erythrocyte membrane
phospholipid composition induced by physical training. In the
present study, there were no significant differences in plasma
cholesterol and triglyceride levels before and after exercise.
Because the lipid status before the exercise may be within the
normal range, it is possible that the effect of the exercise on lipid
metabolism would be blunted.
In contrast, Koga et al.27 measured plasma endogenous digitalis-
like factor (EDLF) before and after physical exercise and reported
that EDLF in exercised hypertensive individuals was significantly
reduced after 10 weeks mild exercise. Our previous report also
demonstrated that the lower membrane fluidity of erythrocytes was
associated with a greater value of plasma EDLF in essential
hypertension.28 Moreover, it was shown that EDLF significantly
increased intracellular free Ca2+.29 Because intracellular Ca2+ has
an important role in the regulation of membrane fluidity,30 the
decrease in plasma EDLF may produce an increase in the
membrane fluidity of erythrocytes. Recently, it has become clear
that ouabain may be the most promising candidate for EDLF,31
although further studies are necessary to determine the role of
EDLF in the regulation of the membrane fluidity of erythrocytes.
One more possible explanation is that nitric oxide (NO) may be
a potential mediator of membrane benefits derived from physical
exercise. Lewis et al.32 demonstrated that exercise increased NO
production from the forearm in hypercholesterolaemic patients and
proposed that NO may play a crucial role in the cardioprotective
effects of exercise. Higashi et al. reported the effects of aerobic
exercise on endothelium-dependent vascular relaxation in hyper-
tensive subjects and demonstrated improvement of the responses in
the exercise group.33 In an in vitro study, we also showed that an
exogenously applied NO donor improved the membrane fluidity of
erythrocytes in patients with essential hypertension, which may
suggest that NO could be a determinant of the membrane fluidity
of erythrocytes in hypertension.34 Additional studies should be
undertaken to assess more thoroughly the relationship between NO
and the exercise-induced changes in membrane function and the
contribution of NO to the beneficial effects of exercise.
Schmidt et al. demonstrated that, after training, the standard
oxygen dissociation curve of haemoglobin was shifted to the right
with a concomitant improvement of all parameters of physical
.
performance (Vo 2max, maximal workload, heart rate during rest and
exercise).35 It can be speculated that the oxygen-binding capacity
of haemoglobin may influence the membrane fluidity of erythro-
cytes during exercise.
In summary, the results of the present study show that aerobic
physical exercise increases the membrane fluidity of erythrocytes
and ameliorates the rigidity of cell membranes in essential hyper-
386 K Tsuda et al.
tensive subjects. Although the mechanisms underlying the
exercise-induced changes of membrane fluidity may be complex
and remain unclear, the improvement of rheological properties of
erythrocytes and microcirculation would partly explain the bene-
ficial effects of physical exercise in hypertension.
ACKNOWLEDGEMENTS
This work was supported, in part, by Grants-in-Aid for Scientific
Research from the Ministry of Education, Science, Sports, Culture
and Technology of Japan (08670819, 10670674), the Naito
Foundation (1993), the Uehara Memorial Foundation (1994 and
1999), the Mitsui Foundation (1999), the Daiwa Health Foundation
(2001) and the Takeda Science Foundation (2002).
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