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This study used ambient heart rate monitoring among health care workers to determine whether a novel measure of heart rate variability (HRV), as well as sleep disturbances, fatigue, or cognitive performance differed among non-rotating night shift nurses relative to those working permanent day shifts. Continuous ambulatory HRV monitoring was performed among night nurses (n = 11), and a comparison group of permanent day nurses (n = 7), over a 36-h period coinciding with the last two 12-h shifts of each participant’s work week. Symptoms and psychomotor vigilance were assessed at the end of the ambient HRV monitoring period, and no differences between shifts were observed. Day nurses exhibited an increase in hourly mean HRV coherence ratios during their sleep period, suggesting a circadian pattern of cardiorespiratory phase coupling, whereas night nurses had no increase in HRV coherence ratios during their sleep period. The HRV coherence patterns were similar to high frequency HRV power among nurses on the same shift. To the authors knowledge, this study was the first to quantify patterns of the HRV coherence ratio among shiftworkers in a non-experimental (work/home) setting. The results suggest a pattern of autonomic dysregulation among night workers during their sleep period relative to those working day shifts. The HRV coherence ratio may serve as a novel indicator of HRV dysregulation among shift workers.