Rotational shifts perturb homeostatic mechanisms in a sexually dimorphic way and may compromise the activity of the autonomic nervous system during day- and night-shifts. Heart rate variability (HRV) is a non-invasive measure to assess autonomic control of the heart. Our aim in this study was to assess HRV by short-term continuous electrocardiogram in female (n=40, average age: 31, average working year: 7) and male (n=40, average age: 29, average working year: 6) nurses under rotational shift programs, HRV is derived from short-term electrocardiogram recordings, carried out both at day- and night -shifts, and included time-domain [e.g., standard deviation of NN intervals, SDNN (ms); percentage of successive RR intervals that differ by more than 50 ms, pNN50 (%); root mean square of successive RR interval differences, RMSSD (ms)] and frequency-domain [very low frequency, VLF; low frequency, LF; high frequency, HF; LF/HF] parameters. Heart rates were similar across the groups but males had lower SDNN (p=0.020), RMSSD (p=0.001). pNN50 (p=0.001), VLF (p=0.048) and HF (p=0.001) but had higher LF/HF ratio (p=0.000) than females. In general, these parameters did not differ between day- and night-shifts (p<0.05). Lower HRV parameters and higher LF/HF in males suggest that they may be under greater threat for disease progression.