Prolonged standing can cause discomfort on the body of the workers and can lead to injury and occupational disease. One of the ergonomic intervention is through improving the work-rest scheduling. The purpose of this study are to identify the fatigue level from the perception of the worker and to investigate the impact of the work-rest scheduling to the standing workers for 12 h working time with a different gender. This study involved two methods which are self-assessment of the worker and direct measurement by using electromyography (EMG). For self-assessment, 80 workers have been interviewed using questionnaire in order to identify the fatigue level. For direct measurement, EMG was attached to the 15 selected workers at their respective leg and lower back to analyse the muscle efforts. In terms of perception, the results show the discomfort and fatigue level at the lower body region in the following order as foot ankle, lower back and leg. There is a significant difference between gender on discomfort pain for foot ankle and leg. The results show short frequent break by 10 min can reduce the fatigue at the leg and infrequent long break is preferable in order to reduce the fatigue at the lower back. In conclusion, it was found that prolonged standing affect the muscle fatigue and discomfort especially lower extremities such as foot ankle, lower back and leg. Besides that, different type of work rest scheduling and gender have significant result towards the muscle fatigue development.