The authors measured 24-h fluid-turnover (FTO) rate during 6 d of preseason training in U.S. college football players. Players, training (T, n = 9, full gear and contact drills) and reference (R, n = 4, conditioning without gear or contact), ingested a deuterium oxide (D2O) dose and provided urine samples every 24 h for analysis of D2O. During one ~2.3–h practice (wet-bulb globe temperature 24.6 °C), body-mass change, urine production, and voluntary fluid intake were measured to calculate gross sweat loss (GSL). Average FTO was 10.3 ± 2.2 L/d for T and 7.0 ± 1.0 L/d for R. GSL was 3.4 ± 1.5 L for T and 1.7 ± 1.3 for R (P > 0.05). By Day 6, body mass decreased significantly in T (–2.4 ± 1.3 kg, P < 0.05) but not in R (0.38 ± 0.95 kg). With preseason training under moderate environmental stress, football players had high FTO and sweat rates, which might have contributed to a loss of body mass during preseason football training.