The purpose of this study was to evaluate the impact of improved hydration on endurance performance and mood in physically active adults. Participants (n = 72; age, 21.0 ± 3.0; 22.2% female) completed two two-mile run trials separated by exactly a week. Before each trial, participants provided a urine sample from the day before the run and a sample from the morning of the run. These samples were analyzed for urine osmolality (UOsm), urine specific gravity (USG), and urine color (Ucol). UOsm and USG levels determined if the participants were placed in either the euhydrated or underhydrated group after the first trial. Those assigned to the euhydrated group were instructed to maintain their current fluid intake levels and those in the underhydrated group were instructed to increase fluid intake levels before the second trial. However, results were grouped by if they improved or maintained their hydration or not. The subjects also completed a Profile of Mood States (POMS) questionnaire before and after each trial to determine mood. Based on conditioning requirements for group assignment, 38% of subjects were classified as underhydrated. There were significant differences between the two trials for both subjects that improved and worsened their hydration in UOsm, USG, Ucol, and thirst (P < 0.05). The group with improved hydration ran -15 ± 67 sec faster in the second trial, while the group that worsened hydration ran 4 ± 26 sec slower in the second trial. When these differences were compared between the two groups with a t-test, there was a trend for statistical differences with a one-way t-test analysis (P = 0.06). When results were split by sex no statistically significant differences were observed (male: -10.8 ± 63.6 sec; female: -29.4 ± 94.8 sec; P > 0.05). Improved hydration did not result in statistically significant difference in TMD or any of the individual mood sub-scales for either group for both males and females (P > 0.05). In conclusion, increased fluid intake to optimize hydration status may affect endurance exercise in young, healthy adults in a two-mile run, but no effect was seen on mood.