Abstract Introduction The gut microbiome plays a crucial role in health and affects mood and behavior. The composition and function of the gut microbiome can be modified by prebiotics, which are defined as substrates selectively utilized by host microorganisms conferring a health benefit. This study examined the effects of a prebiotic diet combination of polydextrose (PDX) and galacto-oligosaccharides (GOS) on cognitive performance and sleepiness during combined sleep restriction and circadian misalignment. Methods Eleven healthy adults (aged 24.6 ± 4.5, 6 females, 5 males) completed a randomized, double-blind, cross-over study lasting a total of 39 days. Participants consumed 14 days of prebiotic (7.5g/day each of PDX [Danisco] and GOS [FrieslandCampina]) or maltodextrin placebo diets at home(sachets dissolved in water with breakfast) while maintaining ~8h sleep per night at habitual times for two separate weeks before undergoing ~4 days of in laboratory testing under combined sleep restriction (3h sleep opportunity per day) and circadian misalignment (wakefulness at night) conditions. Prebiotic or placebo diet continued in-laboratory. Cognitive function testing occurred every 3h during scheduled wakefulness and included the Psychomotor Vigilance Task (PVT), Stroop Color and Word Test (STROOP) visual analog mood scales (VAS), positive and negative affect scales (PANAS), and the Karolinska sleepiness scale (KSS). Participants had a 3-day washout, then repeated the same procedure with the second condition. Results Sleepiness on the KSS was reduced, and positive mood on the PANAS and calm mood were increased under prebiotic versus placebo (all p < 0.05). Median reaction time on the PVT was faster, whereas median reaction times for congruent stimuli on the STROOP task were slower under the prebiotic diet versus placebo (all p < 0.05). Conclusion A prebiotic diet had beneficial effects on many performance, sleepiness and mood outcomes during combined sleep restriction and circadian misalignment. These findings have important implications for shift workers, emergency responders, and military personnel working under the demanding conditions of sleep restriction and circadian misalignment. Support (if any) Office of Naval Research MURI N00014-15-1-2809, NIH CTSA Grant UL1 TR002535 and Undergraduate Research Opportunities Grant, University of Colorado Boulder.