MATERIALS AND METHODS
Twenty-one elite female soccer players who took part in an international soccer competition (July 2001) were subjects of the present study. They were randomly divided into two groups (Table 1). Nine players (18.1 ± 2.3 yr old) in the treatment group received acupuncture treatment. Twelve players (17.7 ± 2.8 yr old) who were not treated acted as the control group. There were no differences in age, body weight, and height and body mass index. All subjects had normal menstrual cycles and did not use oral contraceptives.
Figure 1 shows the experimental design. The subjects were tested over a 3-d period that involved six games. The first game started at 8:30 a.m. and the second started at 2 p.m. on day 1. The third and fourth games (day 2) and fifth and sixth games (day 3) started at same time as day 1. Each game consisted of two 35-min halves, and the playing time of each subject is given in Table 2. The playing time was calculated using a game logbook. After the second game of the day, subjects took a bath, dinner, and rest in their room. Acupuncture treatment was administrated by turns from 8 p.m. to 10 p.m., 1 d before and during the competition. It consisted of placement of disposable 2-gauge stainless needles (40 mm long, SEIRIN, Shizuoka, Japan) at LI 4 (Goukoku) and ST 36 (Ashi-sanri) with 2-Hz electrostimulation (Ohm pulser, Zen Iryoki, Fukuoka, Japan) for 20 min. At the same time, ST 6 (Kyosya), LU 6 (Ko-sai) were also stimulated bilaterally with aurical needles (SEIRIN Jr, SEIRIN) for 15 min during the electroacupuncture treatment (Fig. 2). The general effects of each point include coordination of whole body (LI 4, ST 36), stimulation of salivary gland secretion (LU 6), and treatment and prevention of common cold (ST 6) (Table 3). Acupuncture was performed by a licensed acupuncturist with 10-yr experience in acupuncture treatment.
The study protocol was in accordance with the policy statement of the institutional review board of University of Tsukuba. All subjects had passed a complete medical examination within the past year and received written permission from a specialist sports physician to participate in the study. All subjects submitted informed written consent to the institutional review board. Subjects did not use medications known to affect immune function before and during the study. None of the subjects had allergies or acute infection at the time of the study.
Saliva samples were collected at 6 p.m., 1 wk before (pre), during 4 d (days 1–3) of competition, and 3 d after the competition period (post-3 d). Food intake was prohibited 5 h before salivary collection, but the subjects were free to drink water. Saliva samples were collected as described previously (1). Briefly, the mouth was first rinsed thoroughly with distilled water followed by stimulation of saliva secretion with chewing a sterile cotton swab (Salivette, Sersted, Germany) at a frequency of 120·120 s−1. Saliva was separated from the cotton by centrifugation at 3000 rpm. After measurement of the sample volume, saliva samples were frozen at −80°C and stored until analysis.
Salivary SIgA concentrations were measured by enzyme-linked immunosorbent assay as described previously (1). Briefly, a 96-well microtiter plate (Immulon II, Dynex Technologies, Chantilly, VA) was coated with rabbit antihuman secretory component IgG fraction (MBL, Nagoya, Japan) overnight at 4°C. After adding 250-μL phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA; Sigma, St. Louis, MO), wells were blocked for 2 h. Saliva samples were thawed, centrifuged at 10,000 rpm for 5 min and diluted (1/20) with PBS containing 1% BSA, and 100 μL of each were added and incubated for 1 h. Using purified human SIgA (Organon Teknika, Durham, NC), known concentrations of SIgA were also plated to establish standard values. After washing the plate with PBS-Tween, goat Fab’ anti-IgA conjugated with HRP (MBL) was added to the plate and incubated for 1 h. After washing, substrate solution was added and the color intensity produced after 15 min was measured by a microplate reader (Bio-Rad Laboratories, Hercules, CA) at 490 nm. All samples were assayed in duplicate and the average of absorbance values was used as the representative value. Calibration curves were constructed using standard SIgA concentrations and the concentration of SIgA in the samples was determined from these curves. The intra-assay variability were < 5% on duplicate samples. To avoid interassay variability, all samples from each subject were assayed on the same plate. The interassay coefficient of variation of the method based on analysis of 82 duplicate samples was 6.2%. The concentration of total protein in the saliva was measured by using the method of Bradford (Bio-Rad Laboratories).
Salivary levels of cortisol were analyzed by radioimmunoassay using a commercially available kit (Diagnostic Products Co., Los Angeles, CA). To eliminate interassay variance, all samples were analyzed within the same batch. All intra-assay variances were < 4.9% (15).
Subjective rating of physical well-being.
Various subjective ratings of physical well-being were monitored during the competition. At 6 p.m. of every sampling day, subjects recorded their rating of “muscle tension,” “fatigue,” “lightness of body,” and “flexibility.” These ratings were assessed using a scale of 1 (heavy) to 5 (light).
Profile of mood states (POMS).
A Japanese version of the POMS questionnaire (17) was administrated at 6 p.m. on each sampling day. POMS scores were obtained for subjects on the state of “tension,” “depression,” “anger,” “vigor,” “fatigue,” and “confusion.” Each mood dimension was rated on a scale of 1 to 4 from “not at all” to “extremely.” These subscales appeared to have at least face validity for measuring mood states among athletes. Morgan et al. (19) established the “iceberg profile,” a specific POMS profile that was correlated with success in Olympic qualifying competitions. The “iceberg profile” is characterized by low levels of “tension,” “depression,” “anger,” “fatigue,” and “confusion,” as well as high level of “vigor.”
Data processing and statistical analysis.
For analysis of SIgA levels, data were reported in two forms: (A) absolute concentration of SIgA (μg·mL−1) or (B) SIgA secretion rate (μg·min−1) or the total amount of SIgA in the saliva per unit time. SIgA secretion rate was calculated by multiplying absolute SIgA concentration (μg·mL−1) by saliva flow rate (mL·min−1), which was calculated by dividing the total volume of saliva obtained in each sample (mL) by the time taken to produce the saliva sample (minutes).
Salivary SIgA concentration, saliva flow rate, SIgA secretion rate, salivary cortisol, subjective ratings of physical well-being, and POMS score were analyzed separately by two-way ANOVA with repeated measures to determine the effect of treatment. When ANOVA showed significant effects, a Fisher’s post hoc test was performed. For all analyses, a P-value equal or less than 0.05 was accepted as statistically significant.
Salivary SIgA levels.
The SIgA secretion rate significantly decreased during and after 3 d of competition in the control group (P < 0.05) but did not decrease significantly in the treatment group (Fig. 3). The SIgA secretion rate was significantly higher in the acupuncture group than in the control group at days 1 and 2 (P < 0.05). The concentration of SIgA significantly decreased at day 1 of competition in the control group, but not in the treatment group. In contrast, the SIgA of the treatment group increased at day 3 (Fig. 4).
Salivary cortisol concentrations.
The salivary concentration of cortisol significantly increased at days 2 and 3 in the control group (P < 0.05). In contrast, that in the acupuncture group increased at day 1 (P < 0.05) but returned to baseline at days 2 and 3 (Fig. 5). The salivary concentration of cortisol was significantly lower in acupuncture group than in the control group at days 2 and 3 (P < 0.05). The saliva flow rate and concentration of total protein did not change through the experimental period (data not shown).
Subjective rating of physical well-being.
Compared with the control group, “muscle tension” was significantly lighter at days 1 and 2 in the acupuncture group (P < 0.05, Fig. 6). The score of “lightness of body” was significantly lower at day 3 in the control group than at baseline (P < 0.05) but was not significantly different from the acupuncture group. The score of “fatigue” did not change throughout the study in the acupuncture group, whereas it was heavier during the period of competition in the control group. Furthermore, the score at days 1 and 2 in the acupuncture group was lighter than that in the control group (P < 0.05, P < 0.01, respectively). “Flexibility” score did not change significantly in both groups throughout the study.
In the acupuncture group, “fatigue” remained low during the period of physical competition, whereas the score at day 1 was lower than at baseline (pre, P < 0.05, Fig. 7). In the same group, “confusion” was lower after competition than at baseline (P < 0.05). In the control group, “fatigue” at days 1, 2, and 3 was higher than in the acupuncture group. The POMS score in day 3 was similar with that of day 2 (data not shown). These data suggest that mental fatigue in the control group was higher than in the acupuncture group during the period of sport activity. In contrast, the mental condition as reflected by the POMS showed an iceberg profile in the acupuncture group.
Total playing time in each group.
The total playing time of each subject was given in Table 2. On day 2, the playing time in treatment group was longer than that in control group (P < 0.05).
Our study provides evidence that acupuncture treatment positively affects the salivary SIgA, salivary cortisol, subjective rating of physical well-being and POMS score in elite female soccer players during a relatively short period of sports competition. These results suggest that acupuncture treatment after physical exercise may improve the immunologic and endocrine states of elite female athletes. To our knowledge, the present data are the first to demonstrate that acupuncture treatment inhibits the reduction in mucosal immune function (salivary SIgA levels) induced by intense exercise, and modulates the stress hormone response to physical and competitive stress in elite female athletes.
Our study showed that exercise-induced decrease of salivary SIgA was inhibited in the acupuncture treatment group during the competition period. These data suggest that acupuncture treatment enhances SIgA secretion in the saliva during the period of continuous physical exercise. It is possible that the increased risk of URTI in athletes during the competition period is due to a decrease in SIgA levels. Thus, acupuncture treatment improves SIgA levels and such immunogenic action could lead to a reduced likelihood of infection in athletes and maintenance of physical well-being in female athletes. The exact mechanism of acupuncture-induced improvement of salivary SIgA secretion is not well understood. Sugiyama et al. (28) and Knardahl et al. (13) reported that acupuncture at ST 36 and LI 4 increased muscle sympathetic nerve activity, whereas heart rate, blood pressure, and skin perfusion remained unaffected. Salivary glands are controlled by both sympathetic and parasympathetic nervous systems. Thus, it is possible that acupuncture enhances SIgA secretion by stimulating the sympathetic nervous system. However, our results showed that the resting saliva flow rate did not change during the experimental period. Yang et al. (30) also reported that salivary SIgA increased at 24 h after acupuncture stimulation and continuous treatment of acupuncture significantly increased basal level of SIgA.
Competition differs from training by a greater degree of anxiety and stress that can amplify the hormonal variations usually recorded during exercise (22). Among the hormone reacting to exercise and stress, cortisol is probably the more affected. Increase of plasma as well as salivary cortisol levels during exercise is directly related to the intensity of exercise (5,11). Cortisol also reacts strongly to psychological stress, as during school examination (2). Previous studies demonstrated that intensified training and competition increase plasma cortisol concentration (26,29). In addition, other studies reported enhanced resting cortisol levels in overtrained athletes (12), although resting plasma cortisol levels in overtraining is controversial. Previous studies showed that salivary cortisol levels are correlated to plasma cortisol (27). In our study, the resting salivary cortisol levels increased at days 2 and 3 in the control group. However, acupuncture inhibited exercise-induced increase of salivary cortisol, although no such effect was noted at day 1. There, results suggest that continuous acupuncture treatment modulates the response of cortisol to intense exercise and competitive stress. Although Knardahl et al. (13) reported that plasma cortisol did not change after a single session of acupuncture treatment, competition-induced changes in salivary cortisol might be modulated by acupuncture treatment. A few reports have also shown that acupuncture treatment inhibited the increase of plasma cortisol after surgery (14). It is possible that acupuncture-related stimuli modulate stress response of cortisol.
In a number of studies, self-reported measures such as fatigue, muscle tension, quality of sleep, and other indicators of physical well-being have been monitored to assess adaptation of athletes during intense training and competition period. Miyamoto (18) reported that acupuncture treatment for athletes improved muscle tension and fatigue. In the present study, the acupuncture treatment group reported improvement in most of the subjective ratings of physical well-being, especially “muscle tension” and “fatigue” during the competition period, compared with the control group. Thus, acupuncture treatment improved the perception of physical well-being among elite soccer players.
Mood states have been shown to fluctuate with training volume and intensity (21). Previous studies suggested that mood states such as depression and anxiety may show changes with overtraining (19,21). Previous studies demonstrated significant changes in POMS measures with heavy or increased training (9). Elite athletes score below the general population average on all POMS variables expect vigor, in which they tend to score higher. In the present study, POMS scores suggested a higher mental fatigue in the control group than in the acupuncture group during the competition period. The fall of the fatigue score in the acupuncture group suggests that acupuncture treatment had good effects on mood states during the competition period. Mental well-being was stable in the treatment group showing an iceberg pattern of POMS. Morgan et al. (19) suggested that athletes who are successful exhibit a more pronounced iceberg profile (i.e., better emotional health) than do less successful athletes. Filaire et al. (6) also noted an iceberg profile during successful performance of the soccer team.
It is conceivable that other factors, such as fitness level, might influence the effects of acupuncture treatment on exercise capacity and perception of exertion. Furthermore, it should be recognized that acupuncture is generally practiced within a framework in which the therapy is individualized to the patients and the condition being treated. The present study design, by its very nature of uniformity, is inconsistent with this concept of therapeutic application and therefore may be limited in examining the effectiveness of acupuncture on an individual subject.
Filaire et al. (6) indicated that combined physiological and psychological changes during the training period are primarily of interest when monitoring training stress in relation to physical performance. Finally, studies relating hormonal variations during training and competition are still sparse, mainly because it is difficult to collect biological samples during official sports competition. Measurement of these components in saliva is easy to monitor immunological and hormonal changes to various situations including intense training and competition period.
In this study, the playing time on day 2 differed between groups. The treatment group demonstrated significantly lower cortisol and greater SIgA levels than the control group, although the treatment group played significantly longer than the control group. These data certainly support the positive influence of acupuncture treatment for the athletes of this study.
In the present study, we determined the effect of acupuncture treatment on physical and mental condition of elite female soccer players during sports competition. The following are the main results:
1. Exercise-induced fall in salivary SIgA level was inhibited in the acupuncture group during the competition period.
2. Exercise-induced increase in salivary cortisol was inhibited in the acupuncture group during the competition period.
3. Subjective rating of muscle tension and physical and mental fatigue diminished in the acupuncture group.
4. In the treatment group, POMS score showed an iceberg profile.
These data suggest that acupuncture treatment may improve the physical well-being of female athletes through changes in the immune and endocrine systems.
We thank Keisuke Akimoto (Mitsubishi Heavy Industries, Ltd.) and Dr. Dawei Yang (Tissue Engineering Research Center, National Institute of Advanced Industrial Science and Technology) for critical comment and review of the manuscript.
This study was supported in part by a Grant-in Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (13558003).
1. Akimoto, T., Y. Kumai, T. Akama, et al. Effects of 12 months of exercise training on salivary secretory IgA levels in elderly subjects. Br. J. Sports Med. 37: 76–79, 2003.
2. Allen, P. I., K. A. Batty, C. A. Dodd, et al. Dissociation between emotional and endocrine responses preceding an academic examination in male medical students. J. Endocrinol. 107: 163–170, 1985.
3. Barlas, P., J. Robinson, J. Allen, and G. D. Baxter. Lack of effect of acupuncture upon signs and symptoms of delayed onset muscle soreness. Clin. Physiol. 20: 449–456, 2000.
4. Cook, C. J. Rapid noninvasive measurement of hormones in transdermal exudate and saliva. Physiol. Behav. 75: 169–181, 2002.
5. Cumming, D. C., L. A. Brunsting III, G. Strich, A. L. Ries, and R. W. Rebar. Reproductive hormone increases in response to acute exercise in men. Med. Sci. Sports Exerc. 18: 369–373, 1986.
6. Filaire, E., X. Bernain, M. Sagnol, and G. Lac. Preliminary results on mood state, salivary testosterone: cortisol ratio and team performance in a professional soccer team. Eur. J. Appl. Physiol. 86: 179–184, 2001.
7. Fry, A. C., and W. J. Kraemer. Resistance exercise overtraining and overreaching: neuroendocrine responses. Sports Med. 23: 106–129, 1997.
8. Gleeson, M., W. A. Mcdonald, D. B. Pyne, et al. Salivary IgA levels and infection risk in elite swimmers. Med. Sci. Sports Exerc. 31: 67–73, 1999.
9. Hooper, S. L., L. T. Mackinnon, and S. J. Hanrahan. Mood states as an indication of staleness and recovery. Int. J. Sport Psychol. 23: 1–12, 1997.
10. Karvelas, B. R., M. D. Hoffman, and A. I. Zeni. Acute effects of acupuncture on physiological and psychological responses to cycle ergometry. Arch. Phys. Med. Rehabil. 77: 1256–1259, 1996.
11. Keizer, H., G. M. Janssen, P. Menheere, and G. Kranenburg. Changes in basal plasma testosterone, cortisol, and dehydroepiandrosterone sulfate in previously untrained males and females preparing for a marathon. Int. J. Sports Med. 10: S139–S145, 1989.
12. Kirwan, J. P., D. L. Costill, M. G. Flynn, et al. Physiological responses to successive days of intense training in competitive swimmers. Med. Sci. Sports Exerc. 20: 255–259, 1988.
13. Knardahl, S., M. Elam, B. Olausson, and B. G. Wallin. Sympathetic nerve activity after acupuncture in humans. Pain 75: 19–25, 1998.
14. Kotani, N., H. Hashimoto, Y. Sato, et al. Preoperative intradermal acupuncture reduces postoperative pain, nausea and vomiting, analgesic requirement, and sympathoadrenal responses. Anesthesiology 95: 349–356, 2001.
15. Lac, G., N. Lac, and A. Robert. Steroid assays in saliva: a method to detect plasmatic contaminations. Arch. Int. Physiol. Biochim. Biophys. 101: 257–262, 1993.
16. Mackinnon, L. T., and D. G. Jenkins. Decreased salivary immunoglobulins after intense interval exercise before and after training. Med. Sci. Sports Exerc. 25: 678–683, 1993.
17. Mcnair, D. M., M. Lorr, and L. F. Droppelman. EDITS Manual for the Profile of Mood States. San Diego: Educational and Industrial Testing Service, 1971.
18. Miyamoto, T. Acupuncture treatment for muscle injury [in Japanese]. Jpn. J. Phys. Fitness Sports Med. 43: 39–41, 1997.
19. Morgan, W. P., D. R. Brown, J. S. Raglin, P. J. O’Connor, and K. A. Ellickson. Psychological monitoring of overtraining and staleness. Br. J. Sports Med. 21: 107–114, 1987.
20. O’Connor, P. J., W. P. Morgan, J. S. Raglin, C. M. Barksdale, and N. H. Kalin. Mood state and salivary cortisol levels following overtraining in female swimmers. Psychoneuroendocrinology 14: 303–310, 1989.
21. O’Connor, P. J., W. P. Morgan, and J. S. Raglin. Psychobiologic effects of 3 d of increased training in female and male swimmers. Med. Sci. Sports Exerc. 23: 1055–1061, 1991.
22. Passelergue, P., and G. Lac. Saliva cortisol, testosterone and T/C ratio variations during a wrestling competition and during the post-competitive recovery period. Int. J. Sports Med. 20: 109–113, 1999.
23. Pedersen, B. K., and D. C. Nieman. Exercise immunology: integration and regulation. Immunol. Today 19: 204–206, 1998.
24. Peltham, T. W., L. E. Holt, and R. Stalker. Acupuncture in human performance. J. Strength Cond. Res. 15: 266–271, 2001.
25. Petrovsky, N. Towards a unified model of neuroendocrine-immune interaction. Immunol. Cell Biol. 79: 350–357, 2001.
26. Snegovskaya, V., and A. Viru. Elevation of cortisol and growth hormone levels in the course of further improvement of performance capacity in trained rowers. Int. J. Sports Med. 14: 202–206, 1993.
27. Stupnicki, R., and Z. Obminski. Glucocorticoid response to exercise as measured by serum and salivary cortisol. Eur. J. Appl. Physiol. 65: 546–549, 1992.
28. Sugiyama, Y., Y.-E. Xue, and T. Mano. Transient increase in human muscle sympathetic nerve activity during manual acupuncture. Jpn. J. Physiol. 45: 337–345, 1995.
29. Urhausen, A., and W. Kindermann. Behaviour of testosterone, sex hormone binding globulin (SHBG), and cortisol before and after a triathlon competition. Int. J. Sports Med. 8: 305–308, 1987.
30. Yang, M. M., K. K. Ng, H. L. Zeng, and J. S. Kwork. Effect of acupuncture on immuno globulins of serum, saliva and gingival sulcus fluid. Am. J. Clin. Med. 17: 89–94, 1989.