Objectives: The aim of this study was to compare the psychophysiological strain related to a conventional shift schedule and new ergonomically improved two- and three-shift schedules using heart rate variability (HRV) analysis. The specific aim was to determine whether the introduced ergonomic shift arrangement had any positive effects on the psychophysiological strain such as increased HRV or decrease in the sympathovagal balance of the autonomic nervous system (ANS). Methods: Questionnaire data and 24-hour HRV recordings were gathered from 48 female shift-working nurses once while working the conventional shift schedule (baseline) and again after one year working an ergonomic shift schedule during the morning shift. Results: Comparisons between conventional and ergonomic shift schedules (baseline and follow-up, respectively) revealed significant differences in frequency-domain parameters. Implementing an ergonomic shift schedule resulted in decreased normalized low frequency (LF) power, increased normalized high frequency (HF) power, and decreased LF/HF ratio at the beginning of the shift. Furthermore, at baseline, mean RR interval, root mean square of successive RR interval differences (RMSSD) and normalized HF power were increased at the end of the shift compared with the values at the beginning of the morning shift. In contrast, at the follow-up, LF power was increased between the end and beginning of the morning shift. Conclusions: The psychophysiological strain measured by HRV analysis was lower at the beginning of the work shift for the ergonomic shift schedules compared with the conventional schedule. This indicates that an ergonomic shift schedule may have a positive effect on the ANS recovery occurring between successive work shifts.(J Occup Health 2013; 55: 225-233)