Fatigue associated with shiftwork is a key contributor to human error in the workplace. One way to prevent fatigue-related errors from occurring is to identify fatigue in employees using fitness-for-duty measures. The Occupational Safety Performance Assessment Test (OSPAT), an unpredictable tracking task that measures hand-eye coordination, is currently being used as a fitness-for-duty measure in a variety of industries, but has not yet been validated as a test sensitive to the effects of fatigue. Consequently, the aim of this study was to systematically examine the impact of sustained wakefulness and time-of-day on OSPAT performance. Twenty individuals (10 male, 10 female), aged between 18-25 yr (M = 20.90, SD = 2.29) participated in the study, which was conducted in Australia. The study had a repeated measures design, whereby participants completed the OSPAT and measures of sustained attention (i.e., the psychomotor vigilance task: PVT), and subjective alertness (i.e., the Visual Analog Scale: VAS) every 2 h during 24 h of sustained wakefulness, beginning at 07:00h. Results revealed that VAS ratings of alertness, PVT performance, and OSPAT performance declined significantly as hours of wakefulness increased during the night-time (all p < .01). Furthermore, a positive correlation between OSPAT and PVT performance was observed (r = 0.40, p < .01). Overall, these findings suggest that OSPAT is sensitive to sustained wakefulness during the night-time, and builds the case for OSPAT being a suitable measure for determining fitness-for-duty in workplace environments.