OBJECTIVES: Analysis of HRV has been suggested as a way to study the effects of work-related stresses on cardiovascular autonomic regulation. The aim of this study was to evaluate the use of HRV in the investigation of work-related stressors. METHODS: Cross-sectional data from an ongoing cohort study were used to analyse the relationship of the potential workplace stressors of job-strain, noise and shift work, with HRV. Mean HRV values during sleep and work were calculated in 135 24-h EKG recordings. RESULTS: Shift workers displayed significantly decreased SDNNi levels during sleep, compared with those of the daytime workers (adjusted least square mean values: 69.3 and 85.8 ms, respectively, P < 0.05). Compared with the control group reporting low job demands and high work control (mean: 73.2), we found significantly elevated %LF means during work adjusted for sleep in the low demands, low control group (77.9, P < 0.01), high demands, high control group (77.7, P < 0.05) and high demands, low control group (77.7, P < 0.05). Workers reporting a high noise level compared with a low work noise level also displayed an elevated adjusted mean %LF during work (78.0 and 75.3 respectively, P < 0.06). CONCLUSIONS: The finding of a decreased SDNNi level during sleep in shift workers compared with day workers indicated a less favourable cardiovascular autonomic regulation, which may explain in part the excess cardiovascular disease risk in shift workers. The elevated %LF during work in employees exposed to high job strain or high noise levels indicated a direct shift in the autonomic cardiac balance towards sympathetic dominance. We concluded that the analysis of HRV may provide a useful tool in the study of the physiological effects of work-related stresses.