Abstract (1) Sixteen male subjects participated in resistance training comprising three sets of 8-handgrip exercises at a workload that could be performed no more than eight times, three times a week for 6 weeks. Eight subjects immersed their experimental forearm in cold water (1071 1C) for 20 min following each training period, while the remaining eight served as controls. (2) Muscular endurance with rhythmic handgrips significantly (po0.01) increased in both groups after the training period with a non-significant difference between groups. The relative diameter of ultrasonographyevaluated brachial artery failed to increase in the immersion group despite a significant increase (po0.05) of that in the control group after training. (3) Regular post-exercise cold application might attenuate the improvement in muscular endurance, possibly in association with reduced vascular remodeling. 

H Shiono

M Kosaka

M Yamane

N Ohnishi

N Uchiyama

S Shirasawa

T Okada

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doi:10.1016/j.jth
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E-mail addrJournal of Thermal Biology 29 (2004) 839–843
www.elsevier.com/locate/jtherbioAdaptive changes in muscular performance and circulation by
resistance training with regular cold application
N. Ohnishia,, M. Yamaneb, N. Uchiyamaa, S. Shirasawaa, M. Kosakab,
H. Shionoc, T. Okadac
aDepartment of Human Sciences, Aichi Mizuho College, Hiratobashi, Toyota 470-0394, Japan
bLaboratory for Exercise Physiology and Biomechanics, School of Health and Sport Sciences, Chukyo University,
Kaizu-cho, Toyota 470-0393, Japan
cDepartment of Physiology, Aichi Medical University, Nagakute, Aichi 480-1195, JapanAbstract
(1) Sixteen male subjects participated in resistance training comprising three sets of 8-handgrip exercises at a
workload that could be performed no more than eight times, three times a week for 6 weeks. Eight subjects immersed
their experimental forearm in cold water (1071 1C) for 20min following each training period, while the remaining eight
served as controls. (2) Muscular endurance with rhythmic handgrips significantly (po0.01) increased in both groups
after the training period with a non-significant difference between groups. The relative diameter of ultrasonography-
evaluated brachial artery failed to increase in the immersion group despite a significant increase (po0.05) of that in the
control group after training. (3) Regular post-exercise cold application might attenuate the improvement in muscular
endurance, possibly in association with reduced vascular remodeling.
r 2004 Elsevier Ltd. All rights reserved.
Keywords: Ice treatment; Cryotherapy; Cold; Exercise; Training; Vascular remodeling1. Introduction
Cold application (ice treatment or cryotherapy) is an
effective treatment for acute sports injuries and soft
tissue bruising, acting by decreasing tissue temperature,
blood flow and inflammatory cell invasion of muscle and
thereby inhibiting excessive local inflammatory reactions
(Knight, 1995; Thorlacius et al., 1998). Such treatment
has also been applied to heal exercise-induced damage
following vigorous exercise among athletes. Recently,
cold application has been regularly used to facilitate
recovery from physical and mental fatigue followinge front matter r 2004 Elsevier Ltd. All rights reserve
erbio.2004.08.069
ing author. Tel.: +565-43-0111; fax+565-46-
ess: ohnishi@mizuho-c.ac.jp (N. Ohnishi).exercise in which minimal muscular damage occurs.
Knowledge of the physiological basis for the application
of this treatment to uninjured muscles would be assumed
necessary in order to ensure the effectiveness of athletic
conditioning. However, few studies have evaluated the
effects of regular post-exercise cold application to
uninjured muscles. The authors previously observed that
at 4 weeks of resistance training, improvements in local
muscular endurance were less pronounced with regular
post-exercise application of cold (Teruya et al., 2002).
Although physiological mechanisms to explain our
previous results are unclear, local muscular endurance
might depend on the metabolic capacity of working
muscle cells and/or blood perfusion to working muscles
(Andersen and Henriksson, 1977). It is reported that not
only aerobic exercise training (Baynard et al., 2003;d.
ARTICLE IN PRESS
N. Ohnishi et al. / Journal of Thermal Biology 29 (2004) 839–843840Gavin and Wagner, 2001; Gavin et al., 2004; Laughlin et
al., 2004; Maeda et al., 2001; Miyachi et al., 2001) but
also resistance training (Franke et al., 1998; Rus et al.,
2003), produces vascular growth and increases vasodi-
latory responsiveness. Increased flow-related shear stress
within the vessels and metabolic requirements of skeletal
muscles, including those resulting from tissue hypoxia,
are considered to be the primary factors leading to these
vascular adaptations by inducing the upregulation of
angiogenic growth factors. Moreover, mechanical load-
ing to skeletal muscles is reported to increase local
inflammatory cell infiltration in the presence or absence
of overt injury (McLoughlin et al., 2003; Pizza et al.,
2002). Inflammatory cells are capable of producing
reactive oxygen species, cytokines, and growth factors
that are known to influence skeletal muscle adaptation.
On the other hand, cold application decreases tissue
temperature, blood flow, and metabolic rate. It is
considered that these cold-induced events could decrease
flow-mediated shear stress within the vessels, metabolic
requirements of skeletal muscles and/or neutrophil
invasion of muscle during the recovery period following
exercise, which would affect vascular adaptation in
response to resistance training.
The purpose of this study was to examine the effect of
regular post-exercise cold application on local muscular
endurance and vascular adaptation in response to
resistance training. In addition, plasma interleukin-6
(IL-6, a proinflammatory cytokine) and vascular en-
dothelial growth factor (VEGF, a very potent angio-
genic agent that acts as a specific mitogen for vascular
endothelial cells) were monitored as markers of inflam-
mation and vascular adaptation in response to a bout of
exercise during the training period.2. Methods
2.1. Subjects
Sixteen healthy young male volunteers [age 20.772.3
years, weight 66.278.8 kg and height 173.375.6 cm
(mean7SD)] participated in this study, after providing
written informed consent. Subjects were randomly
assigned to one of two groups.
2.2. Experimental protocol
The subjects performed three sets of eight-handgrip
exercises with their left, non-dominant, hand using a
weight-loaded handgrip ergometer at a that workload
could be performed no more than eight times. Subjects’
right arms served as controls. Participants repeated this
resistance training protocol three times a week for a
total of 6 weeks. Eight of the 16 subjects immersed their
left forearm in cold water for 20min following the lastset of handgrip exercises during each protocol (immer-
sion group). The cold water was stirred and its
temperature was maintained at 1071 1C using a
constant temperature water bath unit (Thermal Robo,
AS ONE Corporation, Osaka, Japan) and cooling unit
(Coolpipe 300-L, TAITEC Co. Ltd., Tokyo, Japan).
The other eight subjects served as controls, remaining in
a sitting position for 20min following training without
immersing their forearm in cold water (control group).
Before and after the 6-week training period, forearm
circumference, maximal handgrip strength, muscular
endurance, and brachial artery diameter in a resting
condition were measured in both arms. Changes of these
measurements in the immersion group were compared
against those in the control group.
IL-6 and VEGF have been known to stimulate
inflammation process and vascular adaptation, respec-
tively. Exercise has been shown to increase Plasma IL-6
and VEGF. In order to examine the possibility that the
cold application might affect exercise-induced adaptive
changes via these processes, plasma IL-6 and VEGF
concentrations were measured before and after the
training protocol at the end of fourth week of the
training period in six subjects in the control group and
seven subjects in the immersion group.
2.3. Measurements
Forearm circumference was measured at the largest
part of the forearm. Maximal handgrip strength was
measured using a digital grip strength meter (ED100N,
Yagami Inc., Nagoya, Japan). Local muscular endur-
ance was evaluated by counting the number of handgrip
repetitions performed every 2 s until the subject could
not tolerate any further contraction at the precise pace
using a weight-loaded handgrip ergometer. A metro-
nome was used to precisely pace the handgrip. Subjects
performed the handgrip exercise in a supine position at a
workload of 30% of maximal pre-training handgrip
strength. Brachial artery diameter was measured by
ultrasonography (EUB-565, Hitachi Medical Corp.,
Tokyo, Japan) in the supine position with the arm in
901 of abduction. The brachial artery was imaged above
the antecubital fossa in the longitudinal plane, with
diameter determined as the distance between the
lumen–intima interfaces of the anterior and posterior
walls.
Plasma IL-6 and VEGF concentrations were mea-
sured by analyzing blood samples taken from the left
antecubital vein before, 30, 60, 90, and 120min after the
handgrip training protocol. Blood samples were with-
drawn into EDTA-containing tubes and centrifuged,
and plasma was stored at 80 1C until analysis. Plasma
concentrations of IL-6 and VEGF were determined by
quantitative sandwich ELISA, using a 96-well microtiter
plate (BioSource International Inc., Camarillo, CA
ARTICLE IN PRESS
AAA
Left (Experimental) arm
cu
la
r 
en
du
ra
nc
e 
(t
im
es
)
100
150
200
250
Pre-training
Post-training
p<0.01
p<0.01
N. Ohnishi et al. / Journal of Thermal Biology 29 (2004) 839–843 841USA). All samples and provided standards were
analyzed in duplicate. An ultra-sensitive kit was used
for IL-6 detection.
2.4. Statistical analysis
All values are reported as mean7SD. Differences
across the testing conditions were determined by two-
way analysis of variance. Significant differences were
accepted at po0.05. Right (Control) arm
Control group Immersion group
Lo
ca
l m
us
Lo
ca
l m
us
cu
la
r 
en
du
ra
nc
e 
(t
im
es
)
0
50
Control group Immersion group
0
50
100
150
200
250
p<0.01
p<0.01
(A)
(B)
Fig. 1. Local muscular endurance of the left (experimental) arm
(A) and the right (control) arm (B) evaluated by counting
repetitions of rhythmic handgrips performed until exhaustion
using a weight-loaded handgrip ergometer before and after 6
weeks of training in the control and the immersion group.
Mean+SD.
r 
of
 b
ra
ch
ia
l a
rt
er
y 
(m
m
)
2
3
4
5
6
Pre-training 
Post-training3. Results
No subjects experienced muscle soreness or injury
during or after the training period. The circumference of
the trained forearm tended to increase after training in
both groups (from 25.071.5 to 25.471.7 cm in the
control group and from 25.371.7 to 25.771.8 cm in the
immersion group) with no significant differences de-
tected. The maximal handgrip strength in the trained
arm did not change significantly in either group over the
training period (from 45.177.9 to 45.979.1 kg in the
control group and from 44.376.4 to 44.676.5 kg in the
immersion group). Exhaustion time for repeated hand-
grip exercise in both arms increased significantly
(po0.01) in the control group and the immersion group
after training (Fig. 1A and B). Increase in that of the
immersion group tended to be smaller as compared with
the control group (nonsignificant difference) after
training. Brachial artery diameter in the trained arm
showed no significant differences before and after
training in both groups (Fig. 2). Left/right (experimental
arm/control arm) ratio for brachial artery diameter in
the control group significantly (po0.05) increased, while
that of the immersion group did not change (Fig. 3).
Plasma concentration of IL-6 from samples collected
at the fourth week of training tended to increase with
time after the 3 sets of handgrip exercise in both groups
(Fig. 4A). In the immersion group, plasma IL-6
concentration was significantly increased at 120min
after exercise compared to pre-exercise levels (po0.05).
No significant changes in plasma VEGF concentration
were observed in response to the handgrip exercise in
either group (Fig. 4B). No significant differences in
plasma IL-6 or VEGF levels were observed between
groups.Control group Immersion group
D
ia
m
et
e
0
1
Fig. 2. Diameter of the brachial artery in a resting condition of
the trained arm measured by ultrasonography before and after
the training period in the control and the immersion group.
Mean+SD.4. Discussion
The 6-week program of resistance training with
regular post-exercise cold application tended to be
shown to inhibit remodeling of the brachial artery. This
coincided with the attenuation of improvement in local
muscular endurance in response to resistance training
ARTICLE IN PRESS
Control group Immersion group
L
/R
 r
a
ti
o
 o
f 
B
ra
c
h
ia
l 
a
rt
e
ry
 d
ia
m
e
te
r
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Pre-training
Post-training
p<0.05
Fig. 3. Left/right (experimental arm/control arm) ratio of the
diameter of the brachial artery measured by ultrasonography
before and after the training period in the control and the
immersion group. Mean+SD.
*
0 30 60 90 120
P
la
sm
a 
co
nc
en
tr
at
io
ns
 (
pg
/m
l)
0
5
10
15
20
25
Time (minutes)
0 30 60 90 120
0
1
2
3
4
5
Control group
Immersion group
 VEGF
 IL-6
(A)
(B)
Fig. 4. Time-related changes in plasma concentration of IL-6
(A) and VEGF (B) determined by quantitative sandwich
ELISA at the end of the fourth week of training in the control
and the immersion group. Mean7SD. *Significantly different
from pre-exercise state (po0.05).
N. Ohnishi et al. / Journal of Thermal Biology 29 (2004) 839–843842observed in our previous study (Teruya et al., 2002) and
in the present study (nonsignificant tendency). A
possible mechanism for these observed changes would
be reduced vascular adaptation due to regular post-
exercise application of cold, leading to attenuated
improvement in muscular endurance. However, the
difference in local muscular endurance between groups
was less pronounced when compared with that seen in
the previous study. In the present study, local muscular
endurance showed a similar tendency to improve even in
control arms that did not undergo training. Because the
subjects in this study were physically untrained, it is
possible that a training-induced increase in centrally
derived motor activity might have contributed to
elevated muscular activity and also obscured the local
effect with cold application.
It is possible that attenuated modification of the
brachial artery in response to training results in cold-
induced vasoconstriction, a decrease in metabolic rate
and/or limitation of neutrophil invasion during the
recovery period. Reduced blood flow due to the
application of cold might decrease biomechanical stress
to vascular endothelial cells during the recovery period,
which might in turn lessen the stimulus for vascular
adaptation. If neutrophil invasion of muscle tissue
decreased with cold application, inflammatory reactions
promoting vascular adaptation might also be inhibited.
However, no physiological evidence was available to
support these speculations, as no significant differences
were observed in plasma IL-6 or VEGF levels in
response to cold application. IL-6 and VEGF levels
have been examined in response to systemic exercise in
many previous studies. As muscle mass influences the
performance of handgrip exercise, it is possible that the
workload employed in the present study was insufficient
to elicit significant change in plasma IL-6 and VEGF
levels. Moreover, it has been reported that neutrophil
invasion (Pizza et al., 2002) and VEGF mRNA
expression (Gavin and Wagner, 2001; Richardson
et al., 2000) in response to exercise, decrease with
advancing exercise training. Further studies under
different experimental conditions, including increased
work load and duration, are needed to ascertain the
behavior of these markers in response to cold applica-
tion during training.
Ice treatment has obvious therapeutic benefits for
tissue in which injury is apparent. However, the present
results indicate the possibility that regular application of
cold, ice treatment or cryotherapy, to uninjured tissue
following exercise might diminish anticipated improve-
ments in physical performance. However, because
athletes who are already well trained might have smaller
reserves for further adaptation, the influence of cold
application might be minor. In addition, the type and
intensity of exercise varies considerably, with exercise
duration tending to be much longer in most training
ARTICLE IN PRESS
N. Ohnishi et al. / Journal of Thermal Biology 29 (2004) 839–843 843programs. Decrease in the exercise-induced stimulus for
adaptation due to application of cold might also be
minor under conditions of longer exercise duration. In
order to apply cryotherapy for conditioning in sports
activities, it is valuable to ascertain any potentially
disadvantageous effects of such therapy. Further studies
are needed to elucidate the mechanisms behind the
possible suppression of improved local muscular endur-
ance and vascular adaptation in response to training
that utilizes application of cold.Acknowledgements
We sincerely thank Dr. Motohiko Miyachi (National
Institute of Health and Nutrition, Japan) for valuable
advice regarding determination of arterial diameter using
ultrasonography. This study was partly supported by a
Grant-in-Aid for Scientific Research from the Ministry of
Education, Science and Culture, Japan (13680065).References
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Adaptive changes in muscular performance and circulation by resistance training with regular cold application

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