Dual energy x-ray absorptiometry (DXA) is a popular tool to determine body composition (BC) in athletes, and is used for analysis of fat-free soft tissue mass (FFST) or fat mass (FM) gain/loss in response to exercise or nutritional interventions. The aim of the current study was to assess the effect of exercise-heat stress induced hypohydration (HYP, >2% of body mass (BM) loss) vs. maintenance of euhydration (EUH) on DXA estimates of BC, sum of skinfolds (SF), and impedance (IMP) measurements in athletes. Competitive athletes (23 males and 15 females) recorded morning nude BM for 7 days before the first main trial. Measurements on the first trial day were conducted in a EUH condition, and again after exercise-heat stress induced HYP. On the second trial day, fluid and electrolyte losses were replaced during exercise using a sports drink. A reduction in total BM (1.6 ± 0.4 kg; 2.3 ± 0.4% HYP) and total FFST (1.3 ± 0.4 kg), mainly from trunk (1.1 ± 0.5 kg), was observed using DXA when participants were HYP, reflecting the sweat loss. Estimated fat percent increased (0.3 ± 0.3%), however, total FM did not change (0.1 ± 0.2 kg). SF and IMP declined with HYP (losses of 1.5 ± 2.9% and 1.6 ± 3% respectively) suggesting FM loss. When EUH was maintained there were no significant changes in BM, DXA estimates, or SF values pre to post exercise, but IMP still declined. We conclude that use of DXA for FFST assessment in athletes must ensure a EUH state, particularly when considering changes associated with nutritional or exercise interventions.