<p>Mental fatigue is a psychobiological state caused by prolonged periods of demanding cognitive activity that affects many aspects of daily life. Two weeks of L-histidine daily ingestion in humans significantly improves mood states compared with a placebo; however, the effects of a single dose are unknown. We aimed to assess the effects of a single dose of L-histidine on mood states, including fatigue, in healthy men and women who regularly experienced fatigue and sleep deprivation. Participants were randomly assigned to the L-histidine or placebo group. They were provided a workload using the Uchida–Kraepelin Performance test, their mood states before and after the workload were assessed, and performance on mental tasks was measured. In the all participants analysis set, the results did not show superiority of the L-histidine group over the placebo group for all endpoints. However, in the subgroup with high fatigue levels, the L-histidine group showed significant decreases compared with the placebo group in the changes in fatigue-inertia (FI), confusion-bewilderment (CB), depression-dejection (DD), and total mood disturbance (TMD) T-scores and a significant increase in the change in vigor-activity (VA) T-score (<i>p</i> = 0.022, 0.049, 0.039, 0.036, and 0.023, respectively). Although a single ingestion of L-histidine showed no significant improvements in any of the endpoints compared with the placebo in the prespecified all-participants analysis set, the exploratory findings suggest that it may alleviate negative mood states, including fatigue, confusion, and depression, and may also improve vigor after a workload in humans with high fatigue levels. Clinical Trial Registry number: UMIN000055719.</p>