The aim of this study was to examine the hypothesis that Japanese female nurses with a reduced total power (TP) component of heart rate variability (HRV) will show an imbalance in sympathovagal activity during night-time work. We carried out this study on 10 healthy female nurses (30.9 ± 3.1 years) working a night shift from 16:00 to 09:00. Heart rate (HR) and the different frequency power components of HRV were measured to estimate individual patterns of sympathovagal activity. A latent imbalance of sympathovagal activity was defined as higher HR and reduced TP, and subjects were divided into two groups based on TP values taken before the night shift. To evaluate central fatigue, we performed three different fatigue tests before and after night work in all subjects. Our results suggest that nurses with reduced HRV have an imbalance of sympathovagal activity but do not suffer from the effects of central fatigue.