Thirst is represented within the anterior cingulate and insular cortices, and may share some common neuroanatomical structures that are implicated with the regulation of mental fatigue. This novel study investigated whether thirst might modulate the subjective, behavioural, or neurophysiological representations of mental fatigue. In a crossover design, thirst was monitored in 15 males during 60 min of cycling in normothermic conditions. Participants either consumed water to the dictates of their thirst (sated), or fluid was withheld and replaced with periodic salt water mouth rinses (thirst). Following either satiety or thirst, a 60 min modified Stroop task was completed to evoke mental fatigue. Prefrontal cortex (PFC) haemodynamics were monitored throughout the prolonged task, and subjective perceptions of fatigue were reported through a visual analogue scale. Behavioural performance was quantified as the total number of Stroop task iterations completed in the mentally fatiguing task, and by collating response time and accuracy into the inverse efficiency score (IES) for each 5 min interval throughout the task. During thirst, fewer iterations were completed and poorer IES performance was evident toward the latter portion of the mentally fatiguing task. Compensatory elevations in PFC oxyhaemoglobin were produced in each condition, however, differed temporally, and were premature during thirst. A diminished capacity to sustain cognitive performance is likely the product of an inability to preserve the distribution of resources within the prefrontal cortex, due to heightened activation about thirst regulatory centres. These data provide novel insight into the relationship between thirst and mental fatigue, and suggest that drinking to the dictates of thirst may be a pertinent strategy to sustain prolonged cognitive performance.