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Sickle cell trait (SCT) is a risk factor of collapse and sudden death in athletes. We conducted a longitudinal study to determine the hematological responses and hydration status in NCAA Division I American football players with SCT. The study took place over 2 years with 6 SCT and 6 position-matched controls (CON) in year 1; and 4 SCT and 4 CON in year 2. In year 2, three of the four SCT players were recruited and re-enrolled with new position-matched controls (total sample data = 10 SCT and 10 CON). Blood samples were taken at three visits: pre-camp, post-camp, and post-season to examine hemoglobin variants, complete blood counts, and chemistry panel 26. Hydration status was assessed by measuring body weight change, urine specific gravity, and urine and sweat electrolyte concentrations during the pre-season training camp. All SCT players were confirmed to have SCT (HbS = 37.9 ± 2.4%) and had greater red cell distribution width (RDW) compared to CON across all visits. Serum uric acid was higher in SCT (7.3 ± 1.0 mg/dL) compared to CON (6.1 ± 0.6 mg/dL; p = 0.001). Furthermore, serum creatine kinase levels were greater in SCT (1617.0 ± 1034.8 IU/L) at pre-camp compared to CON (1037.4 ± 602.8 IU/L; p = 0.03). SCT players exhibited lower pre- and post-practice urine electrolytes and urine specific gravity (SCT pre: 1.019 ± 0.005 vs. CON pre: 1.026 ± 0.008 p < 0.001; SCT post: 1.020 ± 0.005 vs. CON post: 1.030 ± 0.008 p < 0.01), whereas sweat sodium concentrations were higher in SCT players (55.4 ± 13.6 mmol/L) compared to CON (45.5 ± 10.6 mmol/L; p < 0.001). Given the evidence, greater uric acid and CPK levels in SCT players compared to CON may be an early indicator of altered kidney function and muscle damage, which could be added into NCAA guidelines for surveillance among SCT players. Consistent education and reinforcement of the importance of adequate fluid balance during exercise are critical for both SCT and CON players.