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Abstract Introduction Acute total sleep deprivation (TSD) is common due to extended work hours or other time demands. In controlled laboratory settings, individuals exposed to TSD display highly reproduceable profiles of neurobehavioral impairment. In real-world settings, however, TSD is often combined with substance intake, including alcohol and cannabis. In this pilot study, we investigated the effect of substance intake on the neurobehavioral response to TSD. Methods Six healthy individuals (ages 23-37y, all male) completed two 24h in-laboratory study visits separated by ≥1 week. During each visit, they were kept awake from 15:00 until 06:00 the next day, and they completed a 10min PVT every 2-3h. This was followed by an 8h recovery sleep opportunity. For the second visit, participants were randomized – three per group – to receive oral administration of cannabis at 22:30 (10mg) or alcohol at 23:30 (peak blood alcohol concentration of 0.043±0.006% at 00:13, decaying to 0.005±0.007% by 03:55). PVT mean RT, number of lapses (RT>500ms) and false starts were analyzed using mixed-effects regression. Results As expected, PVT performance deteriorated through the second half of the extended waking period (after midnight), with slower mean RT, more lapses, and more false starts (F>3.6, P< 0.002). Alcohol increased mean RT (F=4.9, P=0.002) and lapses (F=7.7, P< 0.001), but did not significantly affect false starts (F=1.0, P=0.41). Cannabis did not significantly affect mean RT (F=0.9, P=0.46), lapses (F=1.1, P=0.37), or false starts (F=0.8, P=0.56) during TSD. Conclusion Alcohol exacerbated neurobehavioral impairment during TSD, even at blood alcohol levels below the legal limit of most countries and states, reaching statistical significance despite our small sample size. Cannabis at the dose provided did not significantly further degrade PVT performance during TSD. Importantly, no attempt was made to make the alcohol and cannabis doses equipotent; therefore, these results should not be interpreted as evidence of their relative effect sizes or safety profiles. Yet, our findings provide preliminary evidence suggesting that commonly used drugs such as alcohol may amplify neurobehavioral impairment from sleep loss, which may have critical implications for automobile driving and other safety-sensitive activities. Support (if any) National Safety Council