Abstract Introduction The Psychomotor Vigilance Task (PVT) is a widely used measure of the effects of sleep deprivation on vigilant attention. The test requires a rapid response to a stimulus occurring at random inter-stimulus intervals (ISIs) between 2 and 10 seconds following a previous response. A few studies have found an ISI effect where shorter ISIs (2-4s) have slower reaction times (RTs) than longer ISIs (8-10s). How this ISI effect is impacted by circadian timing has yet to be investigated. Method This study compared the ISI effect at 3 time points coinciding with 5, 23 (circadian trough), and 29 hours of wakefulness. Data were taken from 16 healthy participants during a 30 hour period of sleep deprivation. Results A repeated-measures (3 x 3) ANOVA (3 time points, 3 ISIs) found that RTs were slower and lapses (RT>500msec) more frequent in short ISIs compared with medium and long ISIs. RTs were also slower and lapses more frequent at the circadian trough compared to both the early testing session and after 29h of total sleep deprivation. Discussion Increased lapses in shorter ISIs could be explained by an involuntary mental “rest period” after responding, that gets longer and more frequent under sleep pressure from both circadian and homeostatic sleep pressure. These findings have implications for interpreting PVT results and their application to everyday tasks, such as driving. Further research could involve neuroimaging the brain’s responses during different ISIs to confirm the mental ‘rest period’ following a response in the PVT task.