Objective: The aim of this study was to evaluate the long-lasting effects of prolonged standing work on a hard floor or floor mat and slow-pace walking on muscle twitch force (MTF) elicited by electrical stimulation. Background: Prolonged standing work may alter lower-leg muscle function, which can be quantified by changes in the MTF amplitude and duration related to muscle fatigue. Ergonomic interventions have been proposed to mitigate fatigue and discomfort; however, their influences remain controversial. Method: Ten men and eight women simulated standing work in 320-min experiments with three conditions: standing on a hard floor or an antifatigue mat and walking on a treadmill, each including three seated rest breaks. MTF in the gastrocnemius-soleus muscles was evaluated through changes in signal amplitude and duration. Results: The significant decrease of MTF amplitude and an increase of duration after standing work on a hard floor and on a mat persisted beyond 1 hr postwork. During walking, significant MTF metrics changes appeared 30 min postwork. MTF amplitude decrease was not significant after the first 110 min in any of the conditions; however, MTF duration was significantly higher than baseline in the standing conditions. Conclusion: Similar long-lasting weakening of MTF was induced by standing on a hard floor and on an antifatigue mat. However, walking partially attenuated this phenomenon. Application: Mostly static standing is likely to contribute to alterations of MTF in lower-leg muscles and potentially to musculoskeletal disorders regardless of the flooring characteristics. Occupational activities including slow-pace walking may reduce such deterioration in muscle function.