ObjectivesThis study aimed to examine the prospective association among objectively measured average working hours (AWHs), frequency of long working hours (FLWHs; defined as ≥205 working hours/month (≥45 hours/week)) for 6 months, and workers’ self-reported psychological and physical health.MethodsThe study included 15 143 workers from 5 Japanese companies. We collected monthly attendance records over 6 months before distributing a questionnaire survey on psychological/physical stress responses and work-related demographics. We then evaluated the associations of those attendance records with psychological/physical measures using analysis of covariance adjusted for sex, age, employment, job type, working conditions, work site and experience of emergency state due to COVID-19.ResultsIrritability, anxiety and depression were significantly greater at ≥180 hours (≥45 hours/week), and fatigue and lack of vigour were greater at ≥205 hours than those of the normal working-hour group (140–180 hours/month [35–45 hours/week]). Psychological indices increased significantly with FLWH, with ≥3 times for irritability, depression and fatigue; ≥2 times for lack of vigour; and ≥1 time for anxiety when compared with no long working hours. No significant associations were observed between AWH or FLWH and physical stress responses.ConclusionsLonger AWH was associated with higher levels of psychological stress responses. The effects of FLWH in the past 6 months varied among the psychological stress responses and did not occur for physical complaints. Under circumstances requiring long hours, workers’ mental health should be protected through minimising the frequency of long work hours.