Vol.2, No.10, 1191-1198 (2010) Health
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Evaluation of autonomic nervous system by heart rate
variability and differential count of leukocytes in
Nobuhiro Suetake1,2*, Yukiko Morita2, Daichi Suzuki3, Keiko Lee1, Hiroyuki Kobayashi1
1Department of Hospital Administration, Juntendo University, School of Medicine, Tokyo, Japan
2Sakae Clinic, Aichi, Japan; *Corresponding Author: nobu666@d1.dion.ne.jp
3Department of Sports Science, Juntendo University, Graduate School of Health and Sports Science, Chiba, Japan
Received 22 May 2010; revised 15 June 2010; accepted 2 July 2010.
Top Japanese sprinters were evaluated for their
physical condition, autonomic function, blood
chemistry, differential leukocyte count and blo-
od lactate level before and after short, maximal
exercise to explore methods of quantifying their
conditioning level. Statistical analysis of data
obtained before and 10 min after exercise were
used to estimate the athletes’ autonomic capac-
ity during recovery. Pre and post exercise varia-
nces in differential leukocyte count revealed str-
ong correlations between neutrophil and symp-
athetic activity, and lymphocyte and parasympa-
thetic activity. The results of the study demon-
strated significant alterations in autonomic pa-
rameters and differential white blood cell count
in response to maximal exercise.
Keywords: Heart Rate Variability; Conditioning;
Differential Count of Leukocytes
It is of paramount importance for high-performance ath-
letes to consistently train in a manner that preserves their
optimal conditioning level. Athletes regularly train at
high intensity and volume for extended periods such that
an imbalance between excessive workload and inade-
quate recovery renders them susceptible to mental and
physical depletion. Therefore, athletes` fitness level is an
important factor in designing training programs and for
preventing overtraining syndrome [1]. However, previ-
ous attempts to portray the blood lactate level and Pro-
file of Mood State (POMS) as respective indicators of
physical and mental fatigue have not been entirely concl-
usive. For this study, heart rate variability (HRV) analy-
sis [2] has been selected to quantitatively evaluate auto-
nomic capacity [3,4] for its greater accuracy in estimat-
ing physical fitness than earlier methods [5]. Cardiac
rhythm is modulated by the two limbs of the autonomic
nervous system (ANS), the sympathetic and parasympa-
thetic, which exert antagonistic effects. Sympathetic do-
minance occurs during stressful conditions such as ner-
vousness and excitement, whereas a shift in favor of va-
gal modulation calms the heart rate. Accordingly, heart
rate and RR interval fluctuation (HRV) permit analogi-
cal inference of autonomic influence as well as the eff-
ects of mental state and stress level on sympathetic and
vagal outputs (HRV analysis) [6,7].
Abo has previously reported that leukocytes are under
autonomic control [8]. More specifically, sympathetic
and parasympathetic stimulations have been shown to
activate adrenalin receptors on granulocytes, the most
prolific leukocytes, and acetylcholine receptors on lym-
phocytes, respectively [9]. Leukocytes have two major
biological defense activities. Granulocytes and macro-
phages incite phagocytic activities against foreign bacte-
ria and materials, whereas lymphocytes respond to viral
and abnormal protein infiltrations by mediating antigen-
antibody production and attacking cellular damage.
In this study we have focused on the proportion of
neutrophil, the representative granulocyte, and lympho-
cyte subpopulations. In short, we have explored whether
the alterations in autonomic parameters and granulo-
cyte/lymphocyte ratio serve as novel markers of physical
and mental exhaustion in sprinters after maximal high
knee lifts, and also act as potential indicators of athletes’
fitness level.
2.1. Subjects
Five elite sprinters in their twenties (mean age 27 ± 1.4)
N. Suetake et al. / Health 2 (2010) 1191-1198
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with qualifications or titles in the Prefectural Track and
Field Championships participated in the study (1 female
and 4 male sprinters with personal bests ranking among
the top 20 in 2009). Weekly training program for the
athletes consisted of daily circuit and weight training for
>/= 3 hr/day for more than 5 days a week, punctuated
with a day of rest and participation in competitive and
time trials on Sundays. All participants routinely en-
gaged in physical exercise for >/= 3 hr/day for at least 5
times per week. Subjects were fully informed about the
objectives, specific test procedures, and mental and ph-
ysical burden of participating in the study. Test times
were scheduled to avoid conflict with competitions and
to prevent carryover effects on athletic performance.
Eligible participants provided full consent before enroll-
ing in the study. Subjects performed a light warm-up
before the test exercise to assess their condition. All st-
udy participants were nonsmokers and had no history of
hypertension, hyperlipidemia, cardiovascular disease, or
diabetes. None of the athletes were taking cardiovascular
medications and psychiatric agents such as sleeping pills
and anxiolytic drugs. On the day of the test, subjects
must have had a body temperature of >/= 37, absence
of significant malaise, >/= 6 hours of sound sleep and
abstinence from alcohol intake for at least 24 hr prior to
the test. Participants were instructed to complete voiding
and avoid food and drink for more than 3 hr before the
scheduled test time. Pre-exercise measurements were
taken in the sitting position after a 20-min rest. Subjects
were monitored to pace their respiration at approxi-
mately 13 breaths/min.
2.2. Method
Heart Rate Variability (HRV) Analysis: By measuring
heart rate and RR interval fluctuations (HRV), autono-
mic activity and the effects of mental balance and stress
level on this capacity can be analogically inferred. RR
interval is the distance between two consecutive R wa-
ves which are known as the largest spikes in EKG. Sp-
ectral transformation of this RR interval variability gene-
rates high frequency power called “HF” (> 0.15 Hz) and
low frequency power termed “LF” (0.04 ~ 0.15 Hz), wh-
ere HF power is regarded as the primary estimate of car-
diac vagal activity. HF power is also generally presumed
to correspond to respiratory sinus arrhythmia and there-
fore approximates to the frequency of respiration. In co-
ntrast, LF power is surmised as mirroring the activities
of the two autonomic nerves. For these reasons, the ratio
of HF and LF is considered as the index of sympathova-
gal activity. The LF/HF ratio is typically associated with
sympathetic activation and HF with vagal excitation. Th-
ese frequency domain indices are obtained from power
spectral analysis that has been calculated from the fast
Fourier transform (FFT) [10].
Primary outcome parameters for this study were freq-
uency domain parameters such as very low frequency
(VLF), high frequency (HF), low frequency (LF), LF/HF
ratio, total power (TP). Additionally, blood chemistry
(AST, ALT, LDH, CK, blood glucose, BUN); differen-
tial WBC count; and blood lactate levels were obtained
before and 10 min after all-out high knee lifts. HF Norm
(adjusted HF) and LF Norm (adjusted LF) were used in
lieu of HF and LF for statistical analyses.
HF Norm = HF/(Total Power VLF) × 100
LF Norm = LF/(Total Power VLF) × 100
The purpose of adjusting the values was to minimize
the impact of VLF alterations and to emphasize the sep-
arate fluctuations induced by parasympathetic and sym-
pathetic activities. VLF has recently garnered attention
as an important prognostic indicator of ischemic heart
disease [11]. Although VLF is considered to reflect the
very slow mechanistic activity of sympathetic control,
the underlying physiological mechanisms remain unclear.
Consequently, VLF measurements were omitted from
this study and the adjusted values were used for statisti-
cal analysis.
Based on the recommendation by The European Soci-
ety of Cardiology and the North American Society of
Pacing and Electrophysiology, resting HRV was meas-
ured with the Heart Rhythm Scanner (HRV analysis sy-
stem from Biocom, Inc.) equipped with software that
performs algorithms of short-term HRV analysis. Meas-
urements were taken from subjects in sitting in a quiet
room before the exercise. Biocom HRM-02 Pulse Wave
Sensor, the photoplethysmography (PPG) monitor used
in the study, was clipped to the right earlobe to measure
HRV for 5 minutes. The same procedure was repeated
after exercise. The PPG monitor was placed above the
converter (light energy) for the light source and pho-
tocells and adjacent to both earlobes to direct the infra-
red beam towards the photocells. Since blood volume
fluctuations within the vasculature of the earlobe corre-
late to beat-to-beat changes, the PPG was employed to
capture the pulse waves to generate data on HRV. At a
designated area of a temperature (24) and humidity
(60%) controlled testing room, participants performed
two sets of 1-min all-out high knee lifts interspersed with
1-min of passive recovery. Tests were conducted be-
tween 12:00 PM – 18:00 PM when HRV was least vari-
able. In order to induce complete exhaustion, a super-
vising trainer provided verbal cues throughout the test
period to monitor and ensure the pace of the exercise.
Participants rested in standing during the 1-min interval
and in supine for 10 minutes during post-exercise recov-
ery. The same pre-exercise autonomic assessment and
blood tests were repeated 10 min after the exercise to
N. Suetake et al. / Health 2 (2010) 1191-1198
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measure and quantitatively evaluate autonomic functions.
Participants were instructed to maintain a consistent res-
piration rate for the autonomic assessment during recov-
ery. Concomitantly with HRV analysis, routine blood
tests for assessing conditioning level have been evalu-
ated in this study, including: 1) blood chemistry aspar-
tate amino transferase (AST), alanine amino transferase
(ALT), lactate dehydrogenase (LDH), creatine kinase
(CK), blood urea nitrogen (BUN) to examine kidney
functions and protein metabolism; 2) blood lactate con-
centration to assess glucose metabolism; and 3) differen-
tial WBC count to investigate recent appraisal of the co-
rrelation between sympathovagal activity and leukocyte
subsets (neutrophils, lymphocytes, monocytes, eosinop-
hils and basophils). Pre and post exercise blood samples
were obtained from the same median cubital vein and
sent to FALCO biosystems Ltd to be tested. Subjects
were immediately asked about their fatigue rating after
the exercise. All laboratory tests and physical measure-
ments were taken while subjects sat calmly in the test
Blood chemistry, differential WBC count, blood lacta-
te level: The following measurements were taken before
and after the stress exercise: 1) blood glucose (BG) to
observe energy expenditure during exercise; 2) aspartate
amino transferase (AST), alanine amino transferase (AL-
T), lactate dehydrogenase (LDH), and creatine kinase
(CK) to evaluate injury/inflammation of muscle tissues;
3) blood urea nitrogen (BUN) to assess alterations in ki-
dney functions and protein metabolism; 4) blood lactate
concentration to determine glucose metabolism; and 5)
differential WBC count (neutrophils, lymphocytes) to
assess autonomic capacity.
2.3. Statistical Analysis
T-test with the level of significance set at P < 0.05 was
used to assess pre and post exercise variations. All sta-
tistical analyses were carried out with PASW Statistics
17.0 (SPSS Inc.) software.
3.1. HRV Analysis (Figures 1 - 5)
The autonomic parameters of all subjects, including total
power (TP), very low frequency (VLF), high frequency
(HF), low frequency (LF) and LF/HF ratio during rest
were all within normal limits.
Figures 1-5 show the comparisons of mean TP, VLF,
LF Norm, HF Norm and LF/HF ratio among the five
Comparisons of mean TP, VLF, LF Norm, HF Norm,
and LF/HF ratio of the five sprinters before and 10 min-
ute after maximal high knee lifts demonstrated a signifi-
cant decrease in TP (p < 0.05), LF Norm(p < 0.01) and
LF/HF ratio (p < 0.05), while a considerable increase in
HF Norm (p < 0.05) was noted. VLF tended towards a
decline despite statistical insignificance (p < 0.1).
Figure 1. Comparison of mean total power among sprinters before
and 10 minute after all-out high knee lifts. A significant decrease
(p < 0.05) was observed after the exercise stress test.
N. Suetake et al. / Health 2 (2010) 1191-1198
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Figure 2. Comparison of mean VLF among sprinters before and
10 minute after all-out high knee lifts. A trend towards decline was
noted after the exercise stress test despite statistical insignificance
(p < 0.1).
Figure 3. Comparison of mean LF Norm among sprinters before
and 10 minute after all-out high knee lifts. A significant decrease
(p < 0.01) was observed after the exercise stress test.
3.2. Blood Chemistry, Blood Lactate
Blood chemistry and blood lactate level were all within
normal limits before and after exercise. All aspects of
blood chemistry of pre and post exercise samples did not
demonstrate significant changes, except for creatine ki-
nase (CK). Creatine kinase (CK) and blood lactate levels
increased significantly 10 minutes after short, maximal
exercise (p < 0.01).
N. Suetake et al. / Health 2 (2010) 1191-1198
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Figure 4. Comparison of mean HF Norm among sprinters before
and 10 minute after all-out high knee lifts. A marked increase (p <
0.05) was observed after the exercise stress test.
Figure 5. Comparison of mean LF/HF ratio among sprinters be-
fore and 10 minute after all-out high knee lifts. A significant de-
crease (p < 0.05) was observed after the exercise stress test.
3.3. Differential Leukocyte Count
(Figures 6-7)
Figures 6-7 illustrate the comparisons of mean neutro-
phil and lymphocyte ratios among the five sprinters be-
fore and after all-out high knee lifts. The proportions of
neutrophils and leukocytes in differential WBC count
were all within normal limits. Neutrophils reduced sig-
nificantly after exercise (p < 0.01), while lymphocytes
increased significantly after exercise (p < 0.01).
N. Suetake et al. / Health 2 (2010) 1191-1198
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Figure 6. Comparison of mean neutrophil count among sprinters
before and 10 minute after all-out high knee lifts. A significant
decrease (p < 0.01) was observed after the exercise stress test.
Figure 7. Comparison of mean leukocyte count among sprinters
before and 10 minute after all-out high knee lifts. A significant in-
crease (p < 0.01) was observed after the exercise stress test.
A battery of blood tests, psychological assessments and
autonomic evaluations are routinely administered to ath-
letes to objectively quantify their conditioning level. The
importance of autonomic capacity in athletic performa-
nce has been elucidated, and reports on autonomic ev-
aluation using HRV analysis in athletes across various
disciplines are available [12]. Despite recent accounts
underpinning the correlation between autonomic activity
N. Suetake et al. / Health 2 (2010) 1191-1198
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and white blood cell count, data on this relationship in
the field of sports and training are practically nonexistent.
Although leukocytosis is a well-established phenomenon
following physical activity, and while reports on differ-
ential WBC alterations in response to exercise have been
published [13], the relationship between pre and post di-
fferential leukocyte and autonomic activity remains equ-
ivocal. The present study focused on quantitative evalu-
ation of pre and post exercise autonomic activities as
well as differential leukocyte count in athletes.
Because the study targeted elite athletes who train
regularly at high intensity and volume, all-out high knee
lifts were selected as the exercise stress test to induce
exhaustion. In the study, a prominent rise in peak lactate
after maximal high knee lifts was noted which served as
an objective marker of exhaustion in participants. Total
power significantly decreased after exercise, but this was
seen as a transient suppression of autonomic functions
under substantial physical stress. Caution must be exer-
cised when training athletes with reduced total power as
this could indicate autonomic dystonia. The association
between autonomic impairment and overtraining syndr-
ome, a chronic state of fatigue, has been reported else-
where. In the present study, significant post-exercise LF
reduction was attributed to the inhibition of baroreceptor
modulation in spite of sympathetic excitation after max-
imal exercise. In fact, LF appeared contingent not only
on sympathetic tone but baroreceptor reflex as well.
In response to a sudden increase in heart rate after sh-
ort maximal workload, parasympathetic tone is thought
to amplify 10 minutes after exercise to counteract the
sympathetic surge. On this basis, post-exercise measure-
ments for this study were taken 10 minutes after brief
maximal load to assess changes during recovery. Modu-
lation of acutely augmented heart rate after all-out exer-
cise has far-reaching ramifications for athletes. The aut-
onomic nervous system maintains physiological homeo-
stasis during abrupt changes in physical states and is sur-
mised to differ significantly between athletes and non-
athletes. In general, it has been suggested that non-athl-
etes have lower parasympathetic tone than athletes. Al-
though this could not be substantiated from this study as
data was obtained from high-performance athletes, ath-
letes and non-athletes are nonetheless expected to dem-
onstrate discrepant autonomic modulations after short
maximal exercise.
One sprinter was excluded from the study due to me-
ntal and physical exhaustion after participating in a 3-
day collegiate track and field competition as the key
player of his team. Resting autonomic tone of this athlete
fell below the normal range and albeit pronounced sub-
jective symptoms, the athlete failed to report his status
prior to the beginning of the study. Moreover, continu-
ous depression of parasympathetic activity was observed
in this athlete. These results suggested that additional
stress load on athletes suffering from overtraining syn-
drome could potentially prolong recovery. Despite rep-
orts of markedly higher vagal tone of athletes than non-
athletes at rest, recovery after exercise also depends lar-
gely on this branch`s activity. In principle, cardiac auto-
nomic assessment using HRV is analogous to the estim-
ation of varying velocity of a moving vehicle from the
coordination of the accelerator and brake. In other words,
HRV is akin to the changing speed of a vehicle, while
the operation of accelerator and brake corresponds to sy-
mpathovagal control of the heart. Because athletes must
rapidly modulate an acutely elevated heart rate during
rest, increasing the vagal tone has important implications.
Our results indicated that LF/HF ratio, an index of sym-
pathetic tone, following maximal high knee lifts declined,
and that HF, an index of parasympathetic function, elev-
ated 10 minutes after exercise. These results were con-
sistent with a shift in proportion of neutrophils and lym-
phocytes in the differential leukocyte count, thus imply-
ing an association between autonomic functions and
white blood cell count. In addition, these findings also
undergirded the potential interactions between sympat-
hetic activity and granulocytes, and parasympathetic ac-
tivity and lymphocytes as cited by Abo [14]. With con-
firmed significant alterations in HRV parameters and
differential leukocyte count in athletes following brief
maximal exercise, quantitative evaluation of autonomic
activity via HRV analysis and differential WBC emerge
as plausible indices of conditioning level and physical
capacity of athletes. The variance in neutrophil and lym-
phocyte ratios appears especially robust as an index, co-
mparable to the assessment of autonomic functions.
Participants of this study involved first-class athletes
who were anticipated to demonstrate superior autonomic
modulation following exercise. In the case of non-ath-
letes, however, post-exercise recovery was expected to
be more sluggish. Since our test date coincided with the
track and field season, recruitment of robust number of
top Japanese athletes for the study was difficult. Several
participants withdrew from the study due to fatigue from
regular training. The sample population fell below the
target size as great care was taken to prevent the physical
burden of participating in the study from ethically inter-
fering with competitions and training programs. More-
over, the nature and intensity of the exercise stress test
were such that only the very best athletes in the country
could perform them adequately, and the selection of
such athletes with comparable athletic capacity resulted
in a maximum sample size of five for this study. Despite
the small sample, this is the first study to evaluate auto-
nomic resilience in sprinters after maximal exercise, and
N. Suetake et al. / Health 2 (2010) 1191-1198
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therefore we believe that our results can serve as a basis
for continued exploration of autonomic modulations in
athletes. While different results may have been observed
had athletes from various disciplines participated in the
study, sprinters were specifically selected because of
their training emphasis on basic exercises and resulting
ability to demonstrate rapid changes in HRV.
According to our results, a greater sample size will
minimize the individual differences in pre and post exer-
cise parameters that were observed in this study. Addi-
tionally, future studies that provide a comparison with a
control group and analyses of graded exercise intensity
and post-exercise intervention will complement our stu-
dy to deepen the understanding of the correlation betw-
een the autonomic nervous system and differential leuk-
ocyte count in athletes.
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