Autonomic Nervous Function During Whole-Body

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sions ( 14,24,29 ), exposures to cold air ( 12 ), or employing local cooling ( 13 ) .... mean(T chest, T scapula, T abdomen) 1 0.14 z mean(T upper arm,. T lower arm) 1 ... freezer until analyzed. ..... Centre for Arctic Medicine at the University of Oulu.


Autonomic Nervous Function During Whole-Body Cold Exposure Before and After Cold Acclimation Tiina M. Mäkinen, Matti Mäntysaari, Tiina Pääkkönen, Jari Jokelainen, Lawrence A. Palinkas, Juhani Hassi, Juhani Leppäluoto, Kari Tahvanainen, and Hannu Rintamäki

MÄKINEN TM, MÄNTYSAARI M, PÄÄKKÖNEN T, JOKELAINEN J, PALINKAS LA, HASSI J, LEPPÄLUOTO J, TAHVANAINEN K, RINTAMÄKI H. Autonomic nervous function during whole-body cold exposure before and after cold acclimation. Aviat Space Environ Med 2008; 79:1–8. Introduction: Cold habituation could affect sympatho-vagal balance, which modulates cold stress responses. The study examined cardiovascular autonomic function at the sinus node level during controlled breathing and while undertaking isometric exercise during whole-body cold exposure before and after cold acclimation. Methods: There were 10 male subjects who were exposed to control (25°C) and cold (10°C) environments for 2 h on 10 successive days in a laboratory. Time and frequency domain heart rate variability (HRV) in terms of root mean square of successive differences in RR intervals, total, high, and low frequency power were determined from controlled breathing at the beginning and end of cold acclimation. Heart rate and blood pressure during an isometric handgrip test (30% MVC for 3 min) were recorded at the beginning and end of cold acclimation. Catecholamines (NE ¯sk), and rectal temperatures (Trect) were measured. and E), mean skin (T Results: Acute cold exposure increased total (36%), low (16%), and high frequency power (25%) and RMSSD (34%). Cold acclimation resulted ¯sk (0.6°C) and lower NE (24%) response in cold. The cold-inin higher T duced elevation in high frequency power became significant after cold acclimation, while other HRV parameters remained unchanged. A smaller increase in heart rate and blood pressure occurred at 10°C during the handgrip test after cold acclimation. Discussion: Cold exposure increased sympathetic activity, which was blunted after cold acclimation. Parasympathetic activity showed a minor increase in cold, which was enhanced after cold acclimation. In conclusion, cold habituation lowers sympathetic activation and causes a shift toward increased parasympathetic activity. Keywords: controlled breathing, habituation, heart rate variability, isometric handgrip.


OLD EXPOSURE IS an environmental stressor that potentially leads to an increase in the loss of body heat. This is counteracted by physiological adjustments that improve thermal insulation from the environment (peripheral vasoconstriction and centralization of circulation) and increase heat production (shivering). The primary role of the sympathetic nervous system during cold exposure is to stimulate peripheral vasoconstriction. This is reflected by an increase in plasma norepinephrine concentrations and blood pressure (30). It is claimed that chronic and repeated cold exposures causing marked whole-body cooling result in more pronounced physiological responses, like enhanced vasoconstriction and metabolic rate (30). However, repeated brief exposures to cold not involving marked whole-

body cooling are suggested to result in habituation. Cold habituation is a form of cold adaptation that denotes the reduction of responses to, or perception of, a repeated stimulation (8). This is suggested to be the most common form of cold adaptation (30). Habituation in humans can develop after only a few repeated brief (, 2 h) exposures to cold air or water (19,21). The observed responses are shivering habituation (e.g., delayed onset and lowered metabolic rate), higher skin temperatures (due to dampened vasoconstriction), less intense cold sensations, and a lowered blood pressure and norepinephrine (NE) response in cold (13,19,30). The diminished rise in blood pressure and plasma NE concentrations suggests that sympathetic activity is affected (18,30). Cardiovascular responses, such as heart rate and blood pressure, have been measured in studies examining cold acclimation due to repeated cold water immersions (14,24,29), exposures to cold air (12), or employing local cooling (13). However, to our knowledge, autonomic nervous system responsiveness measured at the sinus node level by assessing heart rate variability (HRV) while being exposed to cold has not been examined in controlled laboratory conditions. Furthermore, the effects of cold acclimation on HRV are not known. Changes in autonomic nervous system activity assessing HRV and hormonal secretion among over-wintering personnel in Antarctica have been followed previously. Those From the Institute of Health Sciences, University of Oulu, Oulu, Finland; the Finnish Defence Forces, Centre of Military Medicine, Research and Development Unit, Lahti, Finland; the Finnish Defence Forces, Centre of Military Medicine, Aeromedical Centre, Helsinki, Finland; the Department of Physiology, University of Oulu, Oulu, Finland; the Unit of General Practice, Oulu University Hospital, Oulu, Finland; the School of Social Work, University of Southern California, Los Angeles, CA; New Technologies and Risks, Finnish Institute of Occupational Health, Helsinki, Finland; and Physical Work Capacity, Finnish Institute of Occupational Health, Oulu, Finland. This manuscript was received for review in November 2007. It was accepted for publication in June 2008. Address requests for reprints to: Tiina M. Mäkinen, Ph.D., Institute of Health Sciences, University of Oulu, P.O.Box 5000, FI-90014 Oulu, Finland; [email protected] Reprint & Copyright © by the Aerospace Medical Association, Alexandria, VA. DOI: 10.3357/ASEM.2235.2008

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COLD & AUTONOMIC NERVOUS FUNCTION—MÄKINEN ET AL. studies detected decreased sympathetic and increased parasympathetic activity during the residence, followed by a reduction in anterior pituitary and adrenal hormonal secretion (4,11). However, the presence of several concurrent stressors in these isolated environments makes it difficult to distinguish the effects of cold exposure. HRV has also been examined in experiments employing local cooling to the face (16) or using cold pressor tests for evaluating cardiac autonomic function (28). One previous study examined the isolated effects of core and peripheral cooling on HRV and showed that the mode of cooling caused a specific thermoregulatory influence on the very-low frequency component of HRV related to core hypothermia, while skin cooling was associated with less specific HRV changes (5). We consider that studying HRV provides a reliable noninvasive method for the assessment of autonomic cardiovascular regulation and sympatho-vagal interaction. In the present study we also undertook a static isometric contraction test to further examine sympathetic nervous system activity and how it is affected by cold habituation. Both cold exposure and isometric exercise separately involve the activation of the sympathetic nervous system. However, the cardiovascular responses to isometric exercise during whole-body cold exposure are not known. Furthermore, it is not known how cold acclimation affects sympathetic responses during isometric exercise in cold. The objective of the present study was to examine how repeated short cold exposures, mainly causing superficial cooling, are manifested in the cardiovascular regulation by the autonomic nervous system. The study used a cold acclimation protocol known to elicit cold habituation responses (19). Our hypothesis was that cold acclimation would result in cold habituation and changes in HRV, demonstrating a reduction in sympathetic and an increase in parasympathetic activity. METHODS Participants Following approval of the experimental protocol by the University of Oulu and Northern Ostrobothnia Hospital research ethics committee and obtaining written informed consent, 10 young healthy, non-smoking, normotensive male participants volunteered for the study. Mean characteristics of these participants were: age, 22.5 yr (SD 1.6); height, 180.8 m (SD 7.2); weight, 72.4 kg (SD 7.3); body fat, 17.1% (SD 1.9); body mass index, 22.3 kg z m22 (SD 1.6); and peak oxygen con o2max), 53.1 ml z min21 z kg21 (SD 6.1). For sumption (V determining body fat percent, skin fold thickness was measured from triceps, biceps, subscapularis, and suprailiaca and calculated according to Durnin and Rahaman 1967 (3). Maximal oxygen consumption was measured with a bicycle ergometer. The test was ended when the subject could no longer maintain the physical activity level and/or when RQ exceeded 1.15. The  o2max value represented the peak value from the last V 30 s of the test. 2

Experimental Protocol The tests were performed in Oulu, Northern Finland (65 °N 25 °E), during September-November. During this period the mean monthly temperatures ranged between 14.3°C and 20.5°C. The subjects participated in the tests at least 2 h after a meal and were instructed not to use coffee or alcohol the evening before the measurements (, 12 h). They were also instructed to avoid strenuous exercise and to sleep normally (e.g., 8 h) during the night preceding each test. The autonomic tests were performed in a quiet and environmentally controlled laboratory environment. During the experiments the subject were exposed to cold (10°C) for 2 h z d21 on ten successive days. They were lightly clad in shorts, socks, and athletic shoes. The experiments started at the same time each day for each subject. Autonomic nervous function was assessed at the beginning (Day 1) and end (Day 10) of the cold acclimation protocol for both control and cold conditions. Control measurements were performed in a temperature control chamber regulated to ambient air temperature of 25°C. This environment was thermoneutral for the subjects as judged by the measured skin temperatures and thermal sensations. Subjects were then transferred to a second temperature controlled chamber maintained at 10°C for 2 h each day. In both of these climatic chambers the relative humidity was 50 6 3% and air velocity less than 0.2 m z s21. Instrumentation Skin temperatures were measured from 10 sites: forehead, upper back, chest, abdomen, upper arm, lower arm, back of the hand, anterior thigh, dorsal side of the foot, and calf (NTC DC 95, Digi Key, Thief River Falls, MN). The thermistors were attached to the skin with adhesive material. Rectal temperature (Trect) was measured 10 cm beyond the anal sphincter with an YSI 401 probe (Yellow Springs Instrument Co., Yellow Springs, OH). Skin and rectal temperature values were recorded at 1-min intervals with a datalogger (SmartReader 81, ACR Systems, Surrey, BC, Canada) throughout the control and cold exposure. Mean skin temperature (T¯sk) was calculated as an area-weighted average according to the following formula: 0.07 z (Tforehead) 1 0.35 z mean(Tchest, Tscapula, Tabdomen) 1 0.14 z mean(Tupper arm, Tlower arm) 1 0.05 z (Tdorsal hand) 1 0.19 z (Tthigh), 1 0.13 z (Tcalf) 1 0.07 z (Tdorsal side of foot) (10). The presented T¯sk and Trect results represent values recorded during the autonomic nervous system tests. Systolic and diastolic blood pressure was measured from sitting subjects with the arm cuff method immediately before the controlled breathing test using an ambulatory blood pressure monitoring device (Meditech ABPM-04, Meditech Ltd., Budapest, Hungary). Blood pressure was also measured during the sustained handgrip test before the test and in the final minute of the 3 min of sustained exercise. Blood samples (10 ml) were obtained from nine subjects by antecubital venipuncture into EDTA-containing glass tubes (Terumo Venoject, Terumo Corp., Leuven,

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COLD & AUTONOMIC NERVOUS FUNCTION—MÄKINEN ET AL. Belgium) before and after the cold exposure. For technical reasons, no blood sample was available for one subject. Within 10 min, the samples were centrifuged (1500 3 g, 10 min) to harvest the plasma. The plasma was immediately transferred to polypropylene tubes (Eppendorf AG, Hamburg, Germany) and stored in a 280°C freezer until analyzed. The catecholamine analyses consisted of assessing plasma epinephrine (E) and NE concentrations and were performed using the highperformance liquid chromatography method. Autonomic Nervous Function Power spectral analysis allows the quantification of HRV and autonomic responsiveness at different frequency ranges (7). Overall HRV, termed total power (variance of normal-to-normal RR intervals over the selected time segment), is recommended to be divided into three spectral components. These components are integrals from the very low frequency (0–0.04 Hz), low frequency (0.04–0.15 Hz), and high frequency (0.15–0.40 Hz) spectral bands. The distribution of the power and the central frequency of low and high frequency bands are not fixed, but may vary in relation to changes in autonomic modulations of the heart period (26). HRV in the high frequency range is so rapid that it can only be mediated by the parasympathetic nervous system. However, part of the high frequency power seems to be caused by respiration-induced changes in the intrathoracic pressure and blood volume (17). In the low frequency range, both the sympathetic and the parasympathetic nervous system can affect HRV. It seems possible that sympathetic and parasympathetic nervous systems influence each other, and that humoral regulatory systems can also affect the autonomic nervous influences in the HRV. The physiological mechanisms altering HRV in the very low frequency range are not well known. It has been suggested that humoral regulatory mechanisms, thermoregulation, changes in physical activity, and even diurnal variation can influence very low frequency power of HRV (26). HRV during controlled breathing reflects autonomic nervous system regulation at the sinus node level, providing information of sympathetic and parasympathetic activity. This test was performed after 10 min of exposure to 25°C and after 60 min of exposure to 10°C. During the tests the subjects were in supine position and the test was started after a 5-min stabilizing period. Blood pressure was measured before the test using the arm cuff method at the brachialis level. The breathing rate was controlled (0.25 Hz, i.e., 2 s for inspiration and 2 s for expiration) during the study with the use of audio feedback from the data acquisition computer. The duration of the controlled breathing test was 5 min. Electrocardiogram was recorded using a bipolar precordial lead. The recorded signals were digitized with a 12-bit resolution at a sampling rate of 200 Hz (WinAcq-F, Absolute Aliens Oy, Turku, Finland). The analyses were performed offline from stationary regions free of ectopic beats and technical artifacts with WinCPRS software (Absolute Aliens Oy).

Cardiac autonomic function was assessed during the controlled breathing test with 1) time domain analysis of RR interval and 2) power spectral analysis of RR interval. Time domain analysis included the following indices: mean heart rate, root mean square of successive differences (RMSSD) of RR intervals, and the percentage of successive interbeat intervals with over 50-ms differences in duration. In power spectral analysis of HRV, based on Fast Fourier transformation, we determined total power, low-frequency power (0.04–0.15 Hz), and high-frequency power (0.15–0.40 Hz), and low-to-high frequency ratio. Isometric Handgrip Test Static isometric contraction evokes the exercise pressor reflex, which results in vagal withdrawal and sympathetic activation, increasing heart rate, cardiac output, and peripheral vascular resistance during static exercise (1,2,20). This response partially originates in the isometrically contracting muscles, and afferentation from the active muscles to the central nervous system (CNS) is necessary for the development of this peripherally induced response. However, part of this response seems to originate in the CNS without any need for the afferent information from the contracting muscles. This effect is suggested to be related to the CNS activation needed to initiate and maintain the voluntary isometric muscular contraction, the “central command” (1,9). The isometric handgrip test was performed after 20 min of exposure to control (25°C) and after 70 min of exposure to cold (10°C) after the controlled breathing test. During the test the subjects were sitting in a chair in a relaxed position with their arms supported. At first a brief (~3 s) maximal contraction was performed with a dynamometer (Newtest Ltd., Oulu, Finland). After a sufficient recovery period (. 10 min), arm cuff blood pressure and heart rate at rest were recorded. During the test static handgrip was performed with the dominant hand at 30% of maximal voluntary contraction for 3 min. The appropriate level of exercise was monitored from a digital display by the experimenter and subject. The blood pressure and heart rate of the subject were recorded during the last minute of exercise. Statistical Analyses Heart rate, cardiovascular, and temperature parameters between 25°C and 10°C were compared to paired t-tests. When necessary, logarithmic transformations were performed to normalize the data. Data that failed to normalize after logarithmic transformation were analyzed by the nonparametric Wilcoxon sign rank test. Furthermore, the difference in heart rate parameters between cold and warm was calculated, and this difference was compared between Day 1 and Day 10 using the paired t-test (or Wilcoxon sign rank test). For some of the parameters onetailed testing was used. Statistical tests were performed using the SPSS (SPSS 15.0, SPSS Inc., Chicago, IL) software. Data are presented as means 6 SD and the significance was set at P , 0.05.

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COLD & AUTONOMIC NERVOUS FUNCTION—MÄKINEN ET AL. RESULTS At the end of the cold acclimation period the systolic (P 5 0.010) and diastolic blood pressure at 25°C (P 5 0.003) was significantly lower and mean skin temperature (P 5 0.015) was significantly higher than on Day 1. By the end of the cold acclimation period, at 25°C blood pressure was significantly lower (P 5 0.003) than on Day 1 (Table I). There were no significant differences in any of the other parameters (heart rate, NE, E). Mean skin temperature decreased by 6.3–6.4°C (P 5 0.000) and rectal temperature by 0.1–0.2°C during the first hour of exposure to 10°C (P 5 0.044) compared to 25°C. Systolic blood pressure increased on average by 16 mmHg (P 5 0.000-0.001) and diastolic blood pressure by 11 mmHg at 10°C (P 5 0.000-0.022). Plasma NE concentrations were significantly higher (P 5 0.000) and E concentration lower (P 5 0.018) at 10°C compared to 25°C (Table I). Mean skin temperature was 0.3°C higher at 25°C (n.s.) and 0.6°C higher at 10°C (P 5 0.015) at the end of the cold acclimation period (Table I). Systolic blood pressure was 5 mmHg (4%) (P 5 0.010) and diastolic blood pressure 8 mmHg (11%) (P 5 0.003) lower at 25°C on Day 10 compared to Day 1. In addition, the cold acclimation resulted in NE concentrations that were on average 24% lower (P 5 0.043) at 10°C. E was not affected by the cold acclimation period. Exposure to cold increased total power by 36% (P 5 0.028), which indicates a higher overall HRV in cold. Low frequency power was also 16% (P 5 0.033) higher at 10°C. High frequency power was 25% higher at 10°C during Day 1 compared to 25°C (n.s.). RMSSD was 34% (P 5 0.020) longer when measured at 10°C and compared with 25°C (Table II). By the end of the cold acclimation (Day 10), cold exposure increased total power by 51% (P 5 0.040), high frequency power by 54% (P 5 0.043), and RMSSD by 36% (P 5 0.036) in the cold compared to 25°C (Table II). Low-to-high frequency ratio was 10–12% lower at both 25°C and 10°C at the end of the cold acclimation period compared to Day 1 (n.s.). When comparing the change in HRV indices between the end and beginning of the cold acclimation period, no significant differences were observed.

Fig. 1 provides an example of the power spectral density of HRV in one subject during controlled breathing (0.25 Hz) in warm and cold before and after cold acclimation. The figure shows decreased low frequency power (sympathetic activity) both in warm and cold temperature after the cold acclimation period. In addition, in this particular subject the high frequency power is decreased in warm conditions by the end of the cold acclimation. The cardiovascular responses during the 3-min isometric handgrip tests are presented in Table III and Fig. 2. On average, at 25°C heart rate increased by 9–15 bpm (Day 1, P 5 0.001; Day 10, P 5 0.018), systolic blood pressure by 19–23 mmHg (Day 1, P 5 0.000; Day 10, P 5 0.000), and diastolic blood pressure 22–23 mmHg (Day 1, P 5 0.000; Day 10, P 5 0000) during the isometric handgrip. At 10°C heart rate increased by 2–9 bpm (Day 10, n.s.; Day 1, P 5 0.002) systolic blood pressure by 14– 17 mmHg (Day 1, P 5 0.001; Day 10, P 5 0.001) and diastolic blood pressure 12–19 mmHg (Day 10, P 5 0.004; Day 1, P 5 0.000) during the isometric handgrip. By the end of the cold acclimation period the rise in heart rate during the handgrip test was significantly smaller at 10°C compared to Day 1 (P 5 0.015) (Table III). Furthermore, diastolic blood pressure increased less at 10°C on Day 10 compared to Day 1 (P 5 0.035) and also compared to the end of the handgrip test at 25°C (P 5 0.004) (Table III). DISCUSSION Autonomic nervous system responsiveness at the sinus node level (by assessing HRV) in response to cold acclimation has not been previously examined in controlled laboratory conditions. The results of our study confirm earlier research using hormonal and cardiovascular measurements that repeated cold exposure results in a decrease in sympathetic response to acute cold exposure. This is demonstrated by a smaller increase in total HRV, blood pressure, and plasma norepinephrine. The study also demonstrated for the first time an increase in parasympathetic activity following a short period of cold acclimation. This is characterized by a considerable increase in high frequency power after cold acclimation.


Heart rate (bpm) SBP (mmHg) DBP (mmHg) NE (pmol z ml21) E (pmol z ml21) Rectal temperature (°C) Skin temperature (°C)

Day 10







69 6 6 128 6 13 74 6 11 4.6 6 1.9 1.5 6 0.9 37.1 6 0.2 33.1 6 0.6

65 6 11 144 6 14 85 6 7 9.8 6 2.5 1.1 6 0.8 37.0 6 0.2 26.6 6 1.2

ns 0.001 0.022 0.000 0.018 0.044 0.000

68 6 11 123 6 11* 66 6 8 † 4.1 6 1.0 1.2 6 1.1 37.1 6 0.2 33.4 6 0.3

66 6 11 138 6 12 84 6 5 7.4 6 2.3‡ 1.4 6 1.2 36.9 6 0.2 27.2 6 0.7§

ns 0.000 0.000 0.000 ns ns 0.000

The values represent means 6 SD (N 5 10). SBP 5 systolic blood pressure, DBP 5 diastolic blood pressure, NE 5 norepinephrine, E 5 epinephrine. Cold acclimation: significantly different from same exposure during Day 1, * P 5 0.010 (one-tailed), † P 5 0.003 (one-tailed), ‡ P 5 0.043 (one-tailed), § P 5 0.015.


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Total power ms LF ms2 HF ms2 LF/HF (%) RMSSD (ms) pNN50 (%) RRI (ms)

Day 10







5893 6 9144 1518 6 2448 2998 6 5527 101 6 89 77 6 70 31 6 25 882 6 76

9210 6 5307 1813 6 1103 3983 6 3786 87 6 76 117 6 66 50 6 26 962 6 161

0.028 0.033 0.443 0.501 0.020 0.052 0.072

5414 6 5420 1326 6 1376 1826 6 1835 91 6 48 70 6 48 35 6 28 910 6 161

11,011 6 8081 2668 6 2575 4003 6 3993 77 6 47 111 6 63 50 6 20 958 6 205

0.040 0.063 0.043 0.491 0.036 0.104 0.168

The values represent means 6 SD (N 5 10). LF 5 low frequency; HF 5 high frequency; LF/HF 5 low-to-high frequency ratio; RMSSD 5 root mean square of successive differences; pNN50 5 successive interbeat intervals with over 50-ms differences in duration; RRI 5 RR interval.

Consistent with previous research (5), cold exposure significantly increased total and low frequency power of HRV, whereas high frequency power showed an insignificant elevation. The increased low frequency power in cold could indicate increased sympathetic activation, though it is also partially influenced by the parasympathetic activity. Other parameters reflecting sympathetic activation were the increased NE concentrations and higher blood pressure in cold. The observed elevation in

the RMSSD in cold suggests that parasympathetic coactivation also occurs, which response was significantly increased on Day 10, and accompanied by higher HRV in terms of total power in cold. Another indicator supporting increased parasympathetic activity in the cold was the observed slight, but not significant, reduction in heart rate. The observation that both sympathetic and parasympathetic activity is increased during whole-body cooling

Fig. 1. Power spectral density of HRV in one subject during controlled breathing in control (left) and cold (right) conditions during Day 1 (upper panel) and Day 10 (lower panel). Aviation, Space, and Environmental Medicine x Vol. 79, No. 9 x September 2008








70 6 8 78 6 6

85 6 11 87 6 10

0.001 0.018

131 6 5 122 6 9

150 6 5 145 6 13

0.000 0.000

79 6 6 73 6 9

101 6 8 96 6 9

0.000 0.000



77 6 11* 74 6 7†

0.002 0.389

152 6 13† 138 6 10**

166 6 12‡ 155 6 10††

0.001 0.001

92 6 6† 89 6 9†

111 6 9‡‡ 101 6 6

0.000 0.004

68 6 8 72 6 2

Warm (25°C) vs. cold (10°C): * P 5 0.022, † P 5 0.001, ‡ P 5 0.007, **P 5 0.000, †† P 5 0.019, ‡‡ P 5 0.013.

is interesting, as this has not been documented before. It has been suggested that the cooling pattern influences the cardiovascular and autonomic nervous system responses (5,6). Local cooling experiments, e.g., the cold pressor test, caused an increase in sympathetic and a reduction in parasympathetic activity (28), although it must be remembered that the cold pressor responses are mainly related to pain and differ from exposure to cold air. However, on the sinus node level cold-water face immersion elicits increases in cardiac parasympathetic activity (increase in high frequency power), but not in sympathetic activity (16). The study of Fleischer et al. (5) is one of the few examining the relationship between HRV and thermoregulation in response to whole-body cooling in man, although in their study core and peripheral cooling were examined separately. It was observed that core cooling increased the very low frequency power, while very low frequency and low frequency power increased due to skin surface cooling (5). We observed an increase in low frequency power in response to skin cooling, but we also detected an increase in high frequency power at the same time. In the present study, the mean skin temperature was higher and the elevation in plasma NE smaller in the cold after the cold acclimation period. At the same time, a reduction in metabolic rate was observed (22), together with less intense cold sensations than those presented elsewhere (23). These observations are consistent with earlier studies demonstrating habituation to repeated cold stimuli (19,30). Therefore, it was considered that the cold acclimation protocol was successful in eliciting the desired responses. We also observed a reduced blood pressure and increased mean skin temperature in a warm environment after the cold acclimation, which could represent adaptation to the experimental situation with lower sympathetic activity at the beginning of the control test session. A shift in autonomic nervous system activity between the beginning and end of the cold acclimation period was also detected in the present study. This change from the cold-induced elevation in low frequency power on Day 1 to the elevation in high frequency power on Day 10 suggests that at the sinus node level the sympathetic activation due to cold exposure is blunted during cold acclimation, and replaced to some extent by increased 6

parasympathetic influence. This shift in autonomic nervous system activity resembles the results obtained from studies of over-wintering personnel in Antarctica (4,11). The increase in parasympathetic effects at the sinus node level may be due to a true increase in parasympathetic activity. Alternatively, it may also result from reduced sympathetic activity, allowing a better detection of the parasympathetic influence (27). Theoretically, the higher high frequency power by the end of the cold acclimation period could also be due to increased respiratory tidal volume (26). However, during the controlled breathing test the respiratory rate was strictly timed, allowing no significant changes in the tidal volume. Furthermore, tidal volume measurements at rest (results not presented) did not show changes during cold acclimation, supporting the validity of the HRV results during the controlled breathing test. The isometric handgrip test was employed in this study to assess the responsiveness of the autonomic nervous system and its ability to affect the blood pressure responses during the combined effects of cold and isometric exercise. Furthermore, the objective was to examine how a cold acclimation period affects sympathetic responsiveness. Cold exposure and isometric exercise both involve the activation of the sympathetic nervous system. Previous studies examining the effects of local cooling on cardiovascular responses during isometric exercise have recorded additional effects of cold and exercise on the cardiovascular responses (25), whereas others have failed to detect them (15). This is the first study to examine the effects of wholebody cooling on cardiovascular responses during a handgrip test. According to our results, similar elevations in heart rate and blood pressure occurred in both a cold and warm environment before the cold acclimation period. However, cold habituation resulted in a significantly smaller increase in heart rate and diastolic blood pressure during the handgrip test in the cold. No significant differences were observed in the heart rate and blood pressure responses during cold acclimation at 25°C, suggesting that the daily repetitions per se did not influence the responses to isometric handgrip. The lower heart rate and elevations in diastolic blood pressure during isometric handgrip after repeated cold exposures suggest that the sympathetic activation due to

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Fig. 2. Mean difference between start-end of 3-min isometric handgrip test in A) heart rate; B) systolic blood pressure; and C) diastolic blood pressure at 25°C and 10°C. Values represent means 6 SD (N 5 10). Change (start-end) significantly different from Day 1: ‡ P 5 0.015, † P 5 0.0325; significantly different from 25°C: * P 5 0.004.

the handgrip exercise also becomes blunted after cold habituation. Our data do not allow evaluating the role of peripheral and central mechanisms in this change of the isometric handgrip reaction. Further studies are thus needed to assess, e.g., peripheral vascular resistance responses due to isometric exercise in the cold, and would

also provide further insight into cardiovascular control mechanisms. The significance of the results of the present study is that it provides novel information about how repeated cooling and the associated thermal responses reflect the modulation of the autonomic nervous system at the level of the heart. Previous studies have examined the aspect of cold adaptation using classical measures such as hormonal and blood pressure responses (30). In conjunction with these traditional measures, our study examined the independent effects of cold habituation on the sympathetic-vagal balance, using classic tests for assessing autonomic nervous system function. In accordance with our initial hypothesis, this study indicates that cold habituation not only involves a reduction in sympathetic activity, but also increases in parasympathetic activity. We also found that the cardiovascular effects of sympathetic activation due to isometric handgrip were blunted in cold after cold acclimation. This observation emphasizes the possibility that combining training of specific tasks with cold acclimation can lead to physiological changes that are not observed when examined separately. One of the study limitations is that the most marked effects of cold acclimation on the thermal and other physiological responses (e.g., temperature, thermal sensation, blood pressure) could be detected during the initial cooling period (first 30 min of cold exposure). Therefore, performing the tests while also assessing autonomic nervous system function at the beginning of the cooling period might have provided additional information on the effects of cold acclimation. On the other hand, our aim was to assess autonomic activity during a plateau phase after the skin temperatures had stabilized in the cold. It is also recognized that HRV shows day-to-day variation (26), and repeated daily measurements might, therefore, have brought additional information about the successive changes occurring during cold habituation. Furthermore, it cannot be completely excluded that the changing outdoor temperatures during the study period might have partially influenced the physiological responses. Finally, from a statistical perspective, the relatively small sample size may have precluded our ability to have sufficient power to rule out a type II error for some of the responses. In conclusion, whole-body cold exposure involving mainly surface cooling significantly increased blood pressure, plasma NE, total and low frequency power, and RMSSD. Following cold acclimation, mean skin temperature was higher and the increase in plasma NE smaller than before the cold acclimation period. In addition, the increase in high frequency power due to cold exposure became significant after cold acclimation. Cold habituation resulted in diminished sympathetic activity during isometric exercise, as judged by reduced heart rate and diastolic blood pressure elevations during the handgrip test. Our results suggest that cold habituation not only blunts the sympathetic activation due to cold exposure, but also increases the parasympathetic influence, at least at the sinus node level. Further studies ex-

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COLD & AUTONOMIC NERVOUS FUNCTION—MÄKINEN ET AL. amining the successive changes during cold acclimation are warranted. ACKNOWLEDGMENTS This study was partially supported by the Graduate School of Circumpolar Wellbeing, Health and Adaptation coordinated by the Centre for Arctic Medicine at the University of Oulu. Dr. Tiina Mäkinen acknowledges the Finnish Defence Forces for salary support when finalizing this manuscript. We would like to thank the test subjects for their dedication to this study. The experiments performed during this study comply with the current laws of Finland. There are no conflicts of interest in the present study. Authors and affiliations: Tiina M. Mäkinen, Ph.D., Jari Jokelainen, M.Sc., Juhani Hassi, M.D., Ph.D., Institute of Health Sciences, University of Oulu, Oulu, Finland; Tiina M. Mäkinen, Ph.D., Finnish Defence Forces, Centre of Military Medicine, Research and Development Unit, Lahti, Finland; Matti Mäntysaari, M.D., Ph.D., Finnish Defence Forces, Centre of Military Medicine, Aeromedical Centre, Helsinki, Finland; Tiina Pääkkönen, M.Sc., Juhani Leppäluoto, M.D., Ph.D., Hannu Rintamäki, Ph.D., Department of Physiology, University of Oulu, Oulu, Finland; Jari Jokelainen, M.Sc., Unit of General Practice, Oulu University Hospital, Oulu, Finland; Lawrence A. Palinkas, Ph.D., School of Social Work, University of Southern California, Los Angeles, CA; Kari Tahvanainen, M.Sc., New Technologies and Risks, Finnish Institute of Occupational Health, Helsinki, Finland; and Hannu Rintamäki, Ph.D., Physical Work Capacity, Finnish Institute of Occupational Health, Oulu, Finland. REFERENCES 1. Asmussen E. Similarities and dissimilarities between static and dynamic exercise. Circ Res 1981; 48(6, Pt. 2):I3–10. 2. Coote JH, Bothams VF. Cardiac vagal control before, during and after exercise. Exp Physiol 2001; 86:811–5. 3. Durnin JVGA, Rahaman MM. The assessment of the amount of fat in the human body from measurements of skinfold thickness. Br J Nutr 1967; 21:681–9. 4. Farrace S, Ferrara M, De Angelis C, Trezza R, Cenni P, Peri A, et al. Reduced sympathetic outflow and adrenal secretory activity during a 40-day stay in the Antarctic. Int J Psychophysiol 2003; 49(1):17–27. 5. Fleisher LA, Frank SM, Sessler DI, Cheng C, Matsukawa T, Vannier CA. Thermoregulation and heart rate variability. Clin Sci (Lond) 1996; 90:97–103. 6. Frank SM, Raja SN, Bulcao CF, Goldstein DS. Relative contribution of core and cutaneous temperatures to thermal comfort and autonomic responses in humans. J Appl Physiol 1999; 86:1588–93. 7. Freeman R. Assessment of cardiovascular autonomic nervous function. Clin Neurophysiol 2006; 117:716–30. 8. Glossary of terms for thermal physiology, Third edition. Revised by The Commission for Thermal Physiology of the International Union of Physiological Sciences (IUPS Thermal Commission). Jpn J Physiol 2001; 51:245–80. 9. Goodwin GM, McGloskey DI, Mitchell JH. Cardiovascular and respiratory responses to changes in central command during isometric exercise at constant muscle tension. J Physiol 1972; 226:173–90. 10. Hardy JD, DuBois EF, Soderstrom GF. The technic of measuring radiation and convection. J Nutr 1938; 15:461–75. 11. Harinath K, Malhotra AS, Pal K, Prasad R, Kumar R, Sawhney RC. Autonomic nervous system and adrenal responses to cold in man at Antarctica. Wilderness Environ Med 2005; 16:81–91.


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