OBJECT: The purpose of this study was to compare the stress levels of Japanese ambulance men between on-duty and off-duty days, by using the physiological indices of heart rate variability (HRV) and cortisol in urine, measured over each 24-h period. METHODS: Measurements were made during one on-duty and one off-duty day for each subject. The participants were monitored for 24 h with a Holter recording system and a parameter reflecting overall stress levels was obtained by measuring the cortisol level in urine collected over 24 h. RESULTS: The circadian variation of cardiac autonomic nervous system activity was affected when the subjects were on duty. The low-frequency/high-frequency power ratio (=low-frequency power/high-frequency power: LF/HF), which is a useful parameter that reflects the balance of cardiac autonomic nervous activity, differed significantly between the waking and sleeping times on the off-duty day (P=0.03), while it did not differ between these two states on the on-duty day (P=0.56). Similarly, the normalized high-frequency power [=high-frequency/(high-frequency+low-frequency) power: HF/(HF+LF)] ratio, which is a useful measure of the activity of the parasympathetic nervous system, differed significantly between these two states on the off-duty day (P=0.04), while there was no significant difference in the ratio between the two states on the on-duty day (P=0.13). CONCLUSION: These results show that the diurnal balance of the cardiac autonomic nervous system is affected on the on-duty day, even though it is possible for ambulance men to sleep regular hours.