Background: Prolonged physical exercise is known to cause fluid and electrolyte loss through sweating, potentially altering serum biochemical parameters. These changes may impact physiological functions, especially under inadequate hydration. Aim: This study evaluated the effect of physical exercise on serum electrolytes, urea, and creatinine levels in undergraduate footballers in Anambra State, Nigeria. Method: A total of 66 male footballers, divided into three groups of 22 players each, participated in 2-hour football sessions with a 15-minute halftime break. Participants were permitted only 250 mL of water during the session. Blood samples were collected before exercise, within 5 minutes post-exercise, and 2 hours post-exercise. Serum concentrations of sodium, potassium, chloride, bicarbonate, and calcium were determined using ion-selective electrodes. Urea and creatinine levels were analyzed via standard colorimetric methods. Results: There were no statistically significant changes (P > 0.05) in the mean serum concentrations of sodium (Na⁺), potassium (K⁺), chloride (Cl⁻), and bicarbonate (HCO₃⁻) before and after the exercise. However, serum calcium (Ca²⁺) levels increased significantly post-exercise (P < 0.05). Urea and creatinine levels showed no significant variation. Conclusion: The study suggests that short-duration football activity under limited hydration does not significantly alter major serum electrolytes, urea, or creatinine levels, although calcium levels may rise post-exercise. These findings may have implications for hydration and nutritional strategies in athletic training.