Objective: The aims of this study were to determine long-term fatigue effects in the lower limbs associated with standing work and to estimate possible age and gender influences. Background: The progressive accumulation of muscle fatigue effects is assumed to lead to musculoskeletal disorders, as fatigue generated by sustained low-level exertions exhibits long-lasting effects. However, these effects have received little attention in the lower limbs. Method: Fourteen men and 12 women from two different age groups simulated standing work for 5 hr including 5-min seated rest breaks and a 30-min lunch. The younger group was also tested in a control day. Muscle fatigue was quantified by electrically induced muscle twitches (muscle twitch force [MTF]), postural stability, and subjective evaluation of discomfort. Results: MTF showed a significant fatigue effect after standing work that persisted beyond 30 min after the end of the workday. MTF was not affected on the control day. The center of pressure displacement speed increased significantly over time after standing work but was also affected on the control day. Subjective evaluations of discomfort indicated a significant increase in perception of fatigue immediately after the end of standing work; however, this perception did not persist 30 min after. Age and gender did not influence fatigue. Conclusion: Objective measures show the long-term effects of muscle fatigue after 5 hr of standing work; however, this fatigue is no longer perceived after 30 min of rest postwork. Application: The present results suggest that occupational activities requiring prolonged standing are likely to contribute to lower-extremity and/or back disorders.