OBJECTIVE: The aim of this randomized controlled trial (RCT) was to determine whether aerobic exercise during work hours affects cardiac autonomic regulation in cleaners characterized by high levels of occupational physical activity and poor cardiorespiratory fitness. METHOD: Eligible cleaners (n=116) were randomized to an aerobic exercise group (n=59) or a reference group (n=57) with lectures. The intervention group received two 30-min sessions per week of supervised aerobic exercise over 4months. Diurnal measurements of heart rate variability (HRV) and physical activity (accelerometry) were obtained at baseline and at 4-month follow-up. Time and frequency domain indices of HRV were derived during work, leisure time and sleep to evaluate cardiac autonomic regulation. Linear mixed models were used to determine the effect of the intervention on HRV indices, with adjustment for age, gender and daily use of antihypertensive and/or heart medication. RESULTS: Compared with the reference group, the exercise group increased all HRV indices apart from a reduction in LF/HF ratio from baseline to follow-up both during work (p<0.05) and leisure (p<0.05). In contrast, during sleep, the HRV indices tended to decrease in the exercise group compared with the reference group from baseline to follow-up, being significant for the HF spectral component (p=0.03). CONCLUSION: Among cleaners, a worksite aerobic exercise intervention improved cardiac autonomic regulation during work and leisure, but not during sleep. The health effect of this contrasting change in autonomic regulation needs further investigation.