Investigation of driver's physical fitness is an important research topic in accident prevention and intelligent driver monitoring system. Visual attention is one of physical fitness indicators that can be measured quantitatively. Driving simulator can be used to support observation of visual attention and behavior of the driver. On the other side, biomedical effect of driving simulator namely visually induced motion sickness (VIMS) is actively investigated to encourage appropriate usage of the simulator. However, there is no information on how sleep deprivation and day-night difference provoke VIMS and affect visual attention during simulation. In this paper, we present a novel investigation on effect of sleep deprivation and day-night difference on VIMS and visual attention using simulator sickness questionnaire (SSQ) and eye tracking. Statistical analysis on SSQ data shows that day-night difference and sleep deprivation induce symptoms of nausea (F(2,22)=5.825, p < 0.05), oculomotor (F(2,22)=12.657, p < 0.05), and disorientation (F(2,22)=8.270, p < 0.05) significantly. Results of eye tracking analysis show that sleep deprivation and day-night difference affect visual attention significantly (F(2,22)=3.904, p < 0.05). Experimental results suggest that driving simulation is better used in day-time to minimize VIMS. Furthermore, proper night rest beforehand and good room illumination are important to support physical fitness during day-time driving.