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Background In residency programs, it is well known that autonomic regulation is influenced by night duty due to workload stress and sleep deprivation. A less investigated question is the impact on the autonomic nervous system of residents before or when anticipating a night duty shift. In this study, heart rate variability (HRV) was evaluated as a measure of autonomic nervous system regulation. Methods Eight residents in the Department of Anesthesiology were recruited, and 5 minutes of electrocardiography were recorded under 3 different conditions: (1) the morning of a regular work day (baseline); (2) the morning before a night duty shift (anticipating the night duty); and (3) the morning after a night duty shift. HRV parameters in the time and frequency domains were calculated. Repeated measures analysis of variance was performed to compare the HRV parameters among the 3 conditions. Results There was a significant decrease of parasympathetic-related HRV measurements (high-frequency power and root mean square of the standard deviation of R-R intervals) in the morning before night duty compared with the regular work day. The mean difference of high-frequency power between the 2 groups was 80.2 ms (95% confidence interval, 14.5-146) and that of root mean square of the standard deviation of R-R intervals was 26 milliseconds (95% confidence interval, 7.2-44.8), with P = .016 and .007, respectively. These results suggest that the decrease of parasympathetic activity is associated with stress related to the condition of anticipating the night duty work. On the other hand, the HRV parameters in the morning after duty were not different from the regular workday. Conclusions The stress of anticipating the night duty work may affect regulation of the autonomic nervous system, mainly manifested as a decrease in parasympathetic activity. The effect of this change on the health of medical personnel deserves our concern.