Prior studies have utilized heart rate variability (HRV) as the assessment tools for psychological and physiological stress during 24-h shift. However, data regarding effects of prolonged working hours > 24 h on HRV are limited. We aimed to compare between pre- and post-call HRV among physicians who worked 24 plus 8 h. The study included 60 physicians in the internal medicine training. All subjects underwent Holter ECG monitoring for HRV assessment. We compared between HRV of an 8-h regular workday (8am to 4 pm) before on-call duty (pre-call HRV) and an 8-h workday after 24-h on-call duty (post-call HRV). The mean age was 26 +/- 2.5 years. Mean total sleep time during on-call duty was 238.9 +/- 88.3 min. In overall population, the time-domain and frequency-domain HRV parameters were not different between pre- and post-call day. However, the physicians reported their sleep time in the 1st quartile (< 180 min) had significant increase in SDNN, pNN50, high frequency (HF), and decrease in low/high frequency ratio (LF/HF). In contrast, the physicians reported their sleep time in the 4th quartile (> 307.5 min) had significant decrease in pNN50, LF, HF, and increase in heart rate. Multiple linear regression revealed total sleep time as an independent factor associated with pre- and post-call HRV alterations. More sleep during on call (> 5 h) was associated with HRV pattern suggesting both increased sympathetic activity and reduced parasympathetic activity, while less sleep (< 3 h) during on call was associated with post-call parasympathetic rebound HRV pattern.