Objective: The aim of this study was to evaluate the long-lasting motor, behavioral, physiological, and perceptual effects of prolonged standing work in three work–rest cycle conditions including passive or active rest breaks. Background: Muscle fatigue has been evidenced after prolonged standing work through physiological and neuromotor measures. It has been postulated that muscle fatigue induced by prolonged work could be attenuated by appropriate scheduling of work and rest periods. However, investigations in this domain remain limited. Method: Thirty participants simulated standing work for 5 hr with work–rest cycles of short, medium, or long standing periods including passive or active breaks. Lower-leg muscle twitch force (MTF), muscle oxygenation, lower-leg volume, postural stability, force control, and discomfort perception were quantified on 2 days. Results: Prolonged standing induced significant changes in all measures immediately after 5 hr of work, indicating a detrimental effect in long-lasting muscle fatigue, performance, discomfort, and vascular aspects. Differences in the measures were not significant between work cycles and/or break type. Conclusion: Similar physiological and motor alterations were induced by prolonged standing. The absence of difference in the effects induced by the tested work–rest cycles suggests that simply altering the work–rest cycle may not be sufficient to counteract the effects of mainly static standing work. Finally, standing for 3 hr or more shows clear detrimental effects. Application: Prolonged standing is likely to contribute to musculoskeletal and vascular symptoms. A limitation to less than 3 hr of mostly static standing in occupational activities could avoid alterations leading to these symptoms.