To investigate the effects of visual discomfort caused by long-term immersing in virtual environments (VEs), we conducted a comparative study to evaluate users’ visual discomfort in an eight-hour working rhythm and compared the differences between the VEs and the physical environments. Twenty-seven participants performed four different visual tasks with a head-mounted display (HMD) for the VE condition and with a monitor for the physical condition. Their subjective visual discomfort and objective oculomotor indicators were measured to evaluate their visual performances. The results show that the subjective visual fatigue symptoms, the objective pupil size, and the relative accommodation response vary across time for the two conditions, in which VEs affects visual fatigue the most compared to the physical environments. The results also show that pupil size is negatively related to subjective visual fatigue, and the long-term work based on displays only influences the maximum accommodation response of participants. This work is a supplement to the necessary but insufficient-researched field of visual fatigue in long-term immersing in VEs, which should be valuable to researchers involved in the evaluation of visual fatigue using HMDs.