The aim of this study was to investigate differences in heart rate variability (HRV) reflecting the function of autonomic nervous system (ANS) and psycho-physiological strain associated with normal and extended work shifts in nursing work. Complete data were available from 51 female nurses with a mean age of 40 yr, and based on two comparable 36-h HRV measurements supplemented with a questionnaire. Time-domain (meanRR, SDNN, RMSSD) and frequency-domain (LF power, HF power) parameters represented the HRV data, and were analyzed by linear mixed models. The differences between the compared work shifts were minor, revealing mainly increased sympathetic activity at the beginning of the normal work shift. The HRV parameters detected significant differences between work and leisure-time during the normal and extended work shifts in female nurses. During work shifts, an increase in sympathetic and a decrease in parasympathetic control of HRV was observed when compared to the leisure-time situation. Older subjects had overall lower HRV than younger subjects indicating increased sympathetic activation of ANS, especially during work. HRV parameters revealed significant differences between work, leisure-time and sleep of female nurses, but there were few differences between normal and extended work shifts in HRV parameters. This lack of differences between work shifts may be a consequence of the adaptation of nurses to the extended shifts or the more flexible organization of work duties possible during extended work shifts.