Water accounts for 75% of brain mass. Associations may exist between hydration and cognitive performance. The objective of this study was to investigate the effects of dehydration and rehydration on cognitive performance and mood. In this self-control trial, 12 men were recruited from a medical college in Cangzhou, China. After 12 h of overnight fasting, the participants took baseline tests at 8:00 AM on day 2. First morning urine and blood osmolality were analyzed to determine hydration state. Height, weight, and blood pressure were measured following standardized procedures. A visual analog scale for the subjective sensation of thirst was applied, and a profile of mood states questionnaire was applied. Tests were conducted for cognitive performance, including a test of digit span forward and backward, digit-symbol substitutions, dose-work, and stroop effects. Participants were required not to drink water for 36 h but were given three meals on day 3. On day 4, the same indexes were tested as a baseline test. At 8:30 AM, participants drank 1500 mL of purified water over 15 min. After a 1 h interval, the same measurements were performed. Compared with baseline test results, during the dehydration test, participants had lower scores of vigor (11.9 vs. 8.8, %, p = 0.007) and esteem-related affect (8.2 vs. 5.7, %, p = 0.006), lower total scores of digit span (14.3 vs. 13.3, %, p = 0.004), and higher error rates for dose-work (0.01 vs. 0.16, %, p = 0.005). Compared with the dehydration test scores, rehydration test scores showed that fatigue (4.3 vs. 2.1, %, p = 0.005) and total mood disturbance (TMD) (99.0 vs. 90.2, %, p = 0.008) improved, and scores of forward, backward, and total digit span increased (7.7 vs. 8.6, p = 0.014; 5.7 vs. 1.2, p = 0.019; 13.3 vs. 15.4, p = 0.001). Increases were also noted in correct number of digit symbol substitutions, reading speed, and mental work ability (70.8 vs. 75.4, p < 0.001; 339.3 vs. 486.4, n/min, p < 0.001; 356.1 vs. 450.2, p < 0.001), and reaction time decreased (30.2 vs. 28.7, s, p = 0.002). As a conclusion, dehydration had negative effects on vigor, esteem-related affect, short-term memory, and attention. Rehydration after water supplementation alleviated fatigue and improved TMD, short-term memory, attention, and reaction.