The purpose of this study was to examine the effects of two 15-min naps on nurses who work at night in a three-shift system. Of the 15 nurses who were included as study subjects on a night shift, eight took two short naps (the Nap condition), and seven worked without taking a nap (the No-nap condition) during the night shift. We measured sublingual temperature and the bispectral index (BIS), obtained heart rate and heart rate variability measures from an electrocardiogram (ECG), and evaluated sleepiness and fatigue levels every hour using the Visual Analog Scale (VAS). Both subjective sleepiness and fatigue increased between 4:00 and 5:00, with no significant differences observed between the two groups. However, the low- to high-frequency ratio (LF/HF) in the Nap condition group was found to be significantly lower than in the No-nap condition group. Furthermore, a sudden, brief increase in HF values was observed in the No-nap condition group in the morning. The results of this study suggest that taking two short naps may effectively reduce tension and prevent a brief increase in HF values by suppressing sympathetic nervous activity in the morning.