Muscle fatigue, preference and performance of users were explored in this study for several computer mouse control-display gains. Thirty healthy office workers used five different control-display gain settings to complete five sessions of a 30-min Fitts’ task. Muscle activity in the extensor digitorum, extensor carpi ulnaris and flexor digitorum superficialis were measured by surface electromyography (sEMG). Subjective muscle fatigue was assessed using the Borg Rating Scale and user performance was recorded using the GoFitts program. It is found that when the control-display gain was set to 12.97, the highest user effective frequency of operation would be reached. Additionally, subjects reported more subjective muscle fatigue at a control-display gain of 12.97, and sEMG data shows that manipulation would lead to more upper limb muscle fatigue when the control-display gain was set to 8.64 and 12.97. A correlation is found between muscular and subjective fatigue and control-display gains. Similar to subjects’ preference for CDG settings, the commonly used control-display gain settings may result in a high level of fatigue in the forearm muscles. This study found a correlation between muscle fatigue and user preference with control-display gain. Similar to the user preference for control-display gain settings, the control-display gain settings that are commonly used may lead to higher levels of fatigue in the forearm muscles. This study could provide guidance for the selection of a computer mouse control-display gain that can reduce muscle fatigue and fatigue accumulation in office workers who use computer mice.