The aim of this study was to investigate fluctuations in heart rate variability (HRV), which reflect autonomic nervous system (ANS) function and potential psychological and physical strain, among 24 health care workers during work and sleep during four consecutive extended work shifts. Data included 24/36/12 h of HRV measurements, two logbooks, and a questionnaire. A cross-shift/cross-week design was applied. HRV was measured during work, leisure time, and sleep. The HRV data included time-domain [mean RR, SD of normal to normal R-R intervals (SDNN), and root mean square of the successive differences (RMSSD)] and frequency-domain [low frequency (LF)/high frequency (HF) ratio] parameters. HRV parameters revealed significant differences among work, leisure time, and sleep. Mean RR, RMSSD, and SDNN values were lower and the LF/HF ratio was higher on the first versus last day of the work period; however, the differences were most prominent in the morning hours. The results indicate higher levels of cardiovascular stress on the first versus fourth day of the working period, and measurements at night indicate a satisfactory recovery from the extended shifts.