Fatigue can be caused by a lack of quantity and poor quality of sleep due to split sleep. Split sleep is common in train operations because of the limited rest time. This study aims to examine the effect of split sleep on driving fatigue. Fatigue was measured based on the blink duration and frequency through an eye tracker and subjective questionnaires, Karolinska Sleepiness Scale (KSS) and Visual Analogue Scale (VAS). Nine participants underwent two sleep conditions, namely split sleep and consolidated sleep. Participants were asked to drive a train simulator for 2.5 hours in the laboratory after each sleep state was executed. From this study, it was concluded that there were significant differences in the conditions of split sleep and consolidated sleep. This is caused by increased fatigue based on the blink duration and frequency. However, the KSS and VAS data did not show any difference between the two sleep conditions. Therefore, split sleep can be carried out if needed by maintaining the quality and duration of sleep. Improving sleep quality and fulfilling adequate sleep duration (7–8 hours) is carried out as a mitigation strategy to reduce the effect of fatigue due to split sleep. In addition, comfortable lodging in train station can support improving the quality and fulfillment of sleep duration.