Background: Medical residency is a time of high stress and long working hours, which increase the risk of cardiovascular disease. This study aimed to investigate the autonomic modulation of resident physicians throughout the on-call duty cycle. Methods: Spectral analysis of heart rate variability (HRV) was used to compute cardiac parasympathetic modulation (high-frequency power, HF) and cardiac sympathetic modulation (normalized low-frequency power, LF%, and the ratio of LF and HF, LF/HF) of 18 residents for a consecutive 4-day cycle. Results: Male residents show reduced cardiac sympathetic modulation (i.e., higher LF/HF and LF%) than the female interns. Medical residents\' cardiac parasympathetic modulation (i.e., HF) significantly increased on the first and the second post-call day compared with the pre-call day. In contrast, LF% was significantly decreased on the first and the second post-call day compared with the pre-call day. Similarly, LF/HF was significantly decreased on the second post-call day compared with the pre-call day. LF/HF significantly decreased on the first post-call day and on the second post-call day from on-call duty. Conclusion: The guideline that limits workweeks to 80 h and shifts to 28 h resulted in reduced sympathetic modulation and increased parasympathetic modulation during the two days following on-call duty.