To ascertain the usefulness of a 21-item checklist that assesses accumulated fatigue due to overwork, we examined (1) the associations between overtime work, job stressors, or the quantity of sleep/rest and subjective symptoms of fatigue, and (2) whether sleeping hours and monthly days off are associated with the accumulated fatigue parameter using the checklist. Questionnaire surveys were administered twice to Japanese workers at a plant of a manufacturing company. Among the 390 registered workers, 383 workers (284 males and 99 females) in the first survey and 350 workers (260 males and 90 females) in the second survey responded. The subjective symptoms score significantly increased according to the order of grade of overtime work, other job stressors, and sleep/rest subscales in both sexes by ANOVA. The subjective symptoms grade was significantly associated with the other job stressors grade and sleep/rest grade, but not with overtime work. The accumulated fatigue parameter was negatively correlated with daily sleeping hours (significant Spearman's correlation coefficient (r(s)) =-0.318 and -0.340 in the 1st and 2nd surveys) and with monthly days off (r(s)=-0.250 and -0.151) among all of the subjects. It may be possible to assess overwork by the accumulated fatigue parameter.