OBJECTIVE: To compare the circadian variation of hourly heart rate variability (HRV) on work and non-workdays among boilermaker construction workers. METHOD: A panel study of 18 males monitored by 24-h ambulatory ECG over 44 observation-days on paired work and non-workdays was conducted. ECGs were analysed and the SD of normal-to-normal beats index (SDNN(i)) was calculated from 5-min data and summarised hourly. SDNN(i)s over work and non-workdays were compared using linear mixed-effects models to account for repeated measures and harmonic regression to account for circadian variation. RESULTS: Both work and non-work hourly HRV exhibited circadian variation with an increase in the evening and a decrease in the afternoon. SDNN(i) was lower on workdays as compared with non-workdays with the largest, statistically significant differences observed between 10:00 and 16:00, during active working. Lower SDNN(i), albeit smaller yet statistically significant differences, was also observed in the evening hours following work (17:00-21:00) and early morning (4:00). In regression models using all time periods, an average workday SDNN(i) was 8.1 ms (95% CI -9.8 to -6.3) lower than non-workday SDNN(i). The circadian pattern of HRV exhibited two peaks which differed on work and non-workdays. CONCLUSION: While workday and non-workday HRV followed a circadian pattern, decreased HRV and variation of the circadian pattern were observed on workdays. Declines and changes in the circadian pattern of HRV is a concern among this exposed population.