HealthVol.2, No.10, 1191-1198 (2010)
doi:10.4236/health.2010.210175

Evaluation of autonomic nervous system by heart ratevariability and differential count of leukocytes inathletes
Nobuhiro Suetake1,2*, Yukiko Morita2, Daichi Suzuki3, Keiko Lee1, Hiroyuki Kobayashi1
1Department of Hospital Administration Juntendo University School of Medicine, Hongo, Bunkyo-ku, Tokyo, Japan
Sakae Clinic, Nishiki Hotei Bldg.2F, Nishiki, Naka-ku, Nagoya-shi, Aichi, Japan; *Corresponding Author: nobu666@d1.dion.ne.jp
3
Department of Sports Science Juntendo University Graduate School of Health and Sports Science, Hiraga gakuendai, Inba-mura,Inba-gun, Chiba, Japan
2

Received 22 May 2010; revised 15 June 2010; accepted 2 July 2010.

ABSTRACTTop Japanese sprinters were evaluated for their
physical condition, autonomic function, blood
chemistry, differential leukocyte count and blood lactate level before and after short, maximal
exercise to explore methods of quantifying theirconditioning level. Statistical analysis of dataobtained before and 10 min after exercise wereused to estimate the athletes’ autonomic capacity during recovery. Pre and post exercise variances in differential leukocyte count revealed strong correlations between neutrophil and sympathetic activity, and lymphocyte and parasympathetic activity. The results of the study demonstrated significant alterations in autonomic parameters and differential white blood cell countin response to maximal exercise.
Keywords: Heart Rate Variability; Conditioning;
Differential Count of Leukocytes

1. INTRODUCTION
It is of paramount importance for high-performance athletes to consistently train in a manner that preserves theiroptimal conditioning level. Athletes regularly train athigh intensity and volume for extended periods such thatan imbalance between excessive workload and inadequate recovery renders them susceptible to mental andphysical depletion. Therefore, athletes` fitness level is an
important factor in designing training programs and forpreventing overtraining syndrome [1]. However, previous attempts to portray the blood lactate level and Profile of Mood State (POMS) as respective indicators ofphysical and mental fatigue have not been entirely conclusive. For this study, heart rate variability (HRV) analyCopyright © 2010 SciRes.

sis [2] has been selected to quantitatively evaluate autonomic capacity [3,4] for its greater accuracy in estimating physical fitness than earlier methods [5]. Cardiac
rhythm is modulated by the two limbs of the autonomic
nervous system (ANS), the sympathetic and parasympathetic, which exert antagonistic effects. Sympathetic dominance occurs during stressful conditions such as nervousness and excitement, whereas a shift in favor of vagal modulation calms the heart rate. Accordingly, heart
rate and RR interval fluctuation (HRV) permit analogical inference of autonomic influence as well as the effects of mental state and stress level on sympathetic andvagal outputs (HRV analysis) [6,7].
Abo has previously reported that leukocytes are underautonomic control [8]. More specifically, sympathetic
and parasympathetic stimulations have been shown toactivate adrenalin receptors on granulocytes, the mostprolific leukocytes, and acetylcholine receptors on lymphocytes, respectively [9]. Leukocytes have two major
biological defense activities. Granulocytes and macrophages incite phagocytic activities against foreign bacteria and materials, whereas lymphocytes respond to viral
and abnormal protein infiltrations by mediating antigenantibody production and attacking cellular damage.
In this study we have focused on the proportion ofneutrophil, the representative granulocyte, and lymphocyte subpopulations. In short, we have explored whether
the alterations in autonomic parameters and granulocyte/lymphocyte ratio serve as novel markers of physicaland mental exhaustion in sprinters after maximal highknee lifts, and also act as potential indicators of athletes’
fitness level.

2. MATERIALS AND METHODS
2.1. Subjects
Five elite sprinters in their twenties (mean age 27 ± 1.4)
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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 theathletes consisted of daily circuit and weight training for>/= 3 hr/day for more than 5 days a week, punctuatedwith a day of rest and participation in competitive andtime trials on Sundays. All participants routinely engaged in physical exercise for >/= 3 hr/day for at least 5times per week. Subjects were fully informed about theobjectives, specific test procedures, and mental and physical burden of participating in the study. Test times
were scheduled to avoid conflict with competitions andto prevent carryover effects on athletic performance.
Eligible participants provided full consent before enrolling in the study. Subjects performed a light warm-up
before the test exercise to assess their condition. All study participants were nonsmokers and had no history of
hypertension, hyperlipidemia, cardiovascular disease, ordiabetes. 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℃, absenceof significant malaise, >/= 6 hours of sound sleep andabstinence 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 thescheduled test time. Pre-exercise measurements weretaken in the sitting position after a 20-min rest. Subjects
were monitored to pace their respiration at approximately 13 breaths/min.

Fourier transform (FFT) [10].
Primary outcome parameters for this study were frequency 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); differential 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 inlieu of HF and LF for statistical analyses.
HF Norm = HF/(Total Power − VLF) × 100

Heart Rate Variability (HRV) Analysis: By measuring
heart rate and RR interval fluctuations (HRV), autonomic activity and the effects of mental balance and stresslevel on this capacity can be analogically inferred. RR
interval is the distance between two consecutive R waves which are known as the largest spikes in EKG. Spectral transformation of this RR interval variability generates high frequency power called “HF” (> 0.15 Hz) and
low frequency power termed “LF” (0.04 ~ 0.15 Hz), where HF power is regarded as the primary estimate of cardiac vagal activity. HF power is also generally presumed
to correspond to respiratory sinus arrhythmia and therefore approximates to the frequency of respiration. In contrast, LF power is surmised as mirroring the activitiesof the two autonomic nerves. For these reasons, the ratioof HF and LF is considered as the index of sympathovagal activity. The LF/HF ratio is typically associated withsympathetic activation and HF with vagal excitation. These frequency domain indices are obtained from power
spectral analysis that has been calculated from the fast

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 separate fluctuations induced by parasympathetic and sympathetic activities. VLF has recently garnered attention
as an important prognostic indicator of ischemic heartdisease [11]. Although VLF is considered to reflect thevery slow mechanistic activity of sympathetic control,
the underlying physiological mechanisms remain unclear.
Consequently, VLF measurements were omitted fromthis study and the adjusted values were used for statistical analysis.
Based on the recommendation by The European Society of Cardiology and the North American Society ofPacing and Electrophysiology, resting HRV was measured with the Heart Rhythm Scanner (HRV analysis system from Biocom, Inc.) equipped with software that
performs algorithms of short-term HRV analysis. Measurements were taken from subjects in sitting in a quiet
room before the exercise. Biocom HRM-02 Pulse Wave
Sensor, the photoplethysmography (PPG) monitor usedin the study, was clipped to the right earlobe to measure
HRV for 5 minutes. The same procedure was repeatedafter exercise. The PPG monitor was placed above theconverter (light  energy) for the light source and photocells and adjacent to both earlobes to direct the infrared beam towards the photocells. Since blood volume
fluctuations within the vasculature of the earlobe correlate to beat-to-beat changes, the PPG was employed tocapture the pulse waves to generate data on HRV. At adesignated 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 between 12:00 PM – 18:00 PM when HRV was least variable. In order to induce complete exhaustion, a supervising 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 recovery. The same pre-exercise autonomic assessment andblood tests were repeated 10 min after the exercise to

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2.2. Method

N. Suetake et al. / Health 2 (2010) 1191-1198

measure and quantitatively evaluate autonomic functions.
Participants were instructed to maintain a consistent respiration rate for the autonomic assessment during recovery. Concomitantly with HRV analysis, routine blood
tests for assessing conditioning level have been evaluated in this study, including: 1) blood chemistry aspartate 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 concentration to assess glucose metabolism; and 3) differential WBC count to investigate recent appraisal of the correlation between sympathovagal activity and leukocyte
subsets (neutrophils, lymphocytes, monocytes, eosinophils and basophils). Pre and post exercise blood samples
were obtained from the same median cubital vein andsent to FALCO biosystems Ltd to be tested. Subjects
were immediately asked about their fatigue rating after
the exercise. All laboratory tests and physical measurements were taken while subjects sat calmly in the testroom.
Blood chemistry, differential WBC count, blood lactate level: The following measurements were taken before
and after the stress exercise: 1) blood glucose (BG) toobserve energy expenditure during exercise; 2) aspartate
amino transferase (AST), alanine amino transferase (ALT), lactate dehydrogenase (LDH), and creatine kinase(CK) to evaluate injury/inflammation of muscle tissues;3) blood urea nitrogen (BUN) to assess alterations in ki-

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dney functions and protein metabolism; 4) blood lactate
concentration to determine glucose metabolism; and 5)
differential WBC count (neutrophils, lymphocytes) toassess autonomic capacity.

2.3. Statistical Analysis
T-test with the level of significance set at P < 0.05 wasused to assess pre and post exercise variations. All statistical analyses were carried out with PASW Statistics
17.0 (SPSS Inc.) software.

3. RESULTS
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 fivesprinters.
Comparisons of mean TP, VLF, LF Norm, HF Norm,and LF/HF ratio of the five sprinters before and 10 minute after maximal high knee lifts demonstrated a significant decrease in TP (p < 0.05), LF Norm(p < 0.01) andLF/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).

Total Power (ms2/Hz)
Ln
＊

9.00
8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
A
Before Exercise

B
After Exercise

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.
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VLF(ms2/Hz)
Ln
8.00

＊

7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
A
Before Exercise

B
After Exercise

Figure 2. Comparison of mean VLF among sprinters before and
10 minute after all-out high knee lifts. A trend towards decline wasnoted after the exercise stress test despite statistical insignificance(p < 0.1).

LF Norm (n.u.)
Ln
5.00
4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00

A
Before Exercise＊

B
After Exercise

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
Concentration
Blood chemistry and blood lactate level were all within
normal limits before and after exercise. All aspects ofblood chemistry of pre and post exercise samples did notdemonstrate significant changes, except for creatine kinase (CK). Creatine kinase (CK) and blood lactate levels
increased significantly 10 minutes after short, maximal
exercise (p < 0.01).

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LF Norm (n.u.)
Ln
4.50

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119

＊

4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
A
Before Exercise

B
After Exercise

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.

LF/HF ratio
3.50

＊

3.00
2.50
2.00
1.50
1.00
0.50
0.00
A
Before Exercise

B
After Exercise

Figure 5. Comparison of mean LF/HF ratio 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.

3.3. Differential Leukocyte Count (Figures 6- 7)
Figures 6-7 illustrate the comparisons of mean neutrophil and lymphocyte ratios among the five sprinters be-fore and after all-out high knee lifts. The proportions ofneutrophils and leukocytes in differential WBC countwere all within normal limits. Neutrophils reduced significantly after exercise (p < 0.01), while lymphocytes
increased significantly after exercise (p < 0.01).

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Neutrophil（％）
100.00
90.00
80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00

＊

A
Before Exercise

B
After Exercise

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.

Lymphocyte（％）
100.00
90.00
80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00

＊

A
Before Exercise

B
After Exercise

Figure 7. Comparison of mean leukocyte count among sprinters
before and 10 minute after all-out high knee lifts. A significant increase (p < 0.01) was observed after the exercise stress test.

4. DISCUSSION
A battery of blood tests, psychological assessments andautonomic evaluations are routinely administered to athletes to objectively quantify their conditioning level. The

importance of autonomic capacity in athletic performance has been elucidated, and reports on autonomic evaluation using HRV analysis in athletes across various
disciplines are available [12]. Despite recent accounts
underpinning the correlation between autonomic activity

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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 differential WBC alterations in response to exercise have been
published [13], the relationship between pre and post differential leukocyte and autonomic activity remains equivocal. The present study focused on quantitative evaluation of pre and post exercise autonomic activities aswell 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 wasseen as a transient suppression of autonomic functions
under substantial physical stress. Caution must be exercised when training athletes with reduced total power as
this could indicate autonomic dystonia. The association
between autonomic impairment and overtraining syndrome, a chronic state of fatigue, has been reported elsewhere. In the present study, significant post-exercise LF
reduction was attributed to the inhibition of baroreceptor
modulation in spite of sympathetic excitation after maximal 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 short maximal workload, parasympathetic tone is thought
to amplify 10 minutes after exercise to counteract thesympathetic surge. On this basis, post-exercise measurements for this study were taken 10 minutes after brief
maximal load to assess changes during recovery. Modulation of acutely augmented heart rate after all-out exercise has far-reaching ramifications for athletes. The autonomic nervous system maintains physiological homeostasis during abrupt changes in physical states and is surmised to differ significantly between athletes and nonathletes. In general, it has been suggested that non-athletes have lower parasympathetic tone than athletes. Although this could not be substantiated from this study as
data was obtained from high-performance athletes, athletes and non-athletes are nonetheless expected to demonstrate discrepant autonomic modulations after shortmaximal exercise.
One sprinter was excluded from the study due to mental and physical exhaustion after participating in a 3day 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 subjective symptoms, the athlete failed to report his statusprior to the beginning of the study. Moreover, continu-

ous depression of parasympathetic activity was observedin this athlete. These results suggested that additional
stress load on athletes suffering from overtraining syndrome could potentially prolong recovery. Despite reports of markedly higher vagal tone of athletes than nonathletes at rest, recovery after exercise also depends largely on this branch`s activity. In principle, cardiac autonomic assessment using HRV is analogous to the estimation of varying velocity of a moving vehicle from thecoordination 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 sympathovagal control of the heart. Because athletes must
rapidly modulate an acutely elevated heart rate duringrest, increasing the vagal tone has important implications.
Our results indicated that LF/HF ratio, an index of sympathetic tone, following maximal high knee lifts declined,
and that HF, an index of parasympathetic function, elevated 10 minutes after exercise. These results were consistent with a shift in proportion of neutrophils and lymphocytes in the differential leukocyte count, thus implying an association between autonomic functions andwhite blood cell count. In addition, these findings also
undergirded the potential interactions between sympathetic activity and granulocytes, and parasympathetic activity and lymphocytes as cited by Abo [14]. With confirmed significant alterations in HRV parameters anddifferential 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 physicalcapacity of athletes. The variance in neutrophil and lymphocyte ratios appears especially robust as an index, comparable 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-athletes, however, post-exercise recovery was expected tobe 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 thetarget size as great care was taken to prevent the physicalburden of participating in the study from ethically interfering with competitions and training programs. Moreover, 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 ofsuch 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 autonomic resilience in sprinters after maximal exercise, andCopyright © 2010 SciRes.

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therefore we believe that our results can serve as a basis
for continued exploration of autonomic modulations inathletes. 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 exercise parameters that were observed in this study. Additionally, future studies that provide a comparison with acontrol group and analyses of graded exercise intensityand post-exercise intervention will complement our study to deepen the understanding of the correlation between the autonomic nervous system and differential leukocyte count in athletes.

REFERENCES
[1]

[2]

[3]
[4]

[5]

Hedelin, R., Wiklund, U., Bjerle, P., et al. (2000) Cardiac autonomic imbalance in an overtrained athlete.
Medicine and Science in Sports and Exercise, 32(9),1531-1533.
Hayano, J., Sakakibara, Y., Yamada, A. and Mukai, S.
(1991) Accuracy of assessment of cardiac vagal tone byheart rate variability in normal subjects. American Journal of Cardiology, 67(2), 199-204.
Tanabe, S., Terao, T. and Nakano, S. (1993) Sympathovagal balance in young athletes. Tokai Journal ofSports Science and Medicine, 5, 44-49.
Tanabe, S., Terao, T. and Nakano, S. (1995) Comparisonof sympathovagal balance between the middle aged andyoung athletes. Tokai Journal of Sports Science andMedicine, 7, 75-82.
Perini, R. and Veicsteinas, A. (2003) Heart rate variability and autonomic activity at rest and during exercise in

Copyright © 2010 SciRes.

various physiological conditions. European Journal ofApplied Physiology, 90(3-4), 317-325.
[6] Sawada, Y. (1999) Heart rate variability, Is it available inpsychophysiological research. Japanese Journal of Cancer Research, 26, 8-13.
[7] Yokoi, M. and Yamazaki, K. (1995) Cardiac autonomic
nervous activity interaction under mental stress. A study
using the corrected heart rate variability indexes. Tokai
Journal of Sports Science and Medicine, 7, 75-82.
[8] Abo, T. (2002) Autonomic nervous regulation of leukocytes. Japanese Journal of Physical Fitness and Sports
Medicine, 51, 10-11.
[9] Abo, T. (1999) Regulation of leukocytes by the autonomic nervous system, new rule on the cooperation ofneuro-endocrine-immune systems (on the functions of
the autonomic nervous system). Japanese Journal of
Psychosimatic Medicine, 39, 67-74.
[10] Goto, K., Matuura, H. and Muramoto, K. (2002) Estimate of autonomic nervous system function by heart ratevariability analysis [in Japanese] IEICE technical report.
ME and Bio Cybernetics, 102(507), 13-16.
[11] Hadase, M., Azuma, A. and Zen, K. (2004) Very low
frequency power of heart rate variability is a powerfulpredictor of clinical prognosis in patients with congestive
heart failure. Japanese Circulation Society, 68(4), 343347.
[12] Tanabe, S., Yoshioka, K. and Yamashita, Y. (1994)
Sympathetic and parasympathetic activities at rest andafter exercise in judo athletes. Tokai Journal of Sports
Science and Medicine, 6, 35-42.
[13] Suzuki, K., Saoto, H. and Endo, T. (1995) Variation ofneutrophil active oxygen and differential count of leukocyte’s during maximal exercise load in athletes. Japanese
Journal of Physical Fitness and Sports Medicine, 44(6),747.
[14] Abo, T. (2002) Number and function of leukocytes are
regulated by the autonomic nervous system. Journal ofInternational Society of Life Information Science, 20(1),171-189.

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