This cross-sectional comparative study investigated the levels of insomnia, anxiety, and heart rate variability of nurses members working different shift systems. One hundred and twenty-four participants were recruited from members of the nurses of two Taiwanese hospitals. Data were collected using the Chinese versions of the Athens Insomnia Scale and Beck Anxiety Scale questionnaires and electrocardiograms recorded immediately upon completion of each participant's work shift. A binary logistic regression model was used for analysis. Insomnia, anxiety, and abnormal parasympathetic activity were more acute in nurses who worked a rotating shift than in those performing day or night shift work. Logistic regression analysis showed that age significantly increased the incidence and level of insomnia. Age, years of service, and nurses' status as a parent significantly intensified incidences of anxiety and abnormal parasympathetic activity. Rotating shift work is one of the main factors causing adverse effects on the physical and psychological health of nurses; therefore, when a shift work system cannot be avoided, a practice of day and night shifts for nurses is preferable to rotating shifts.