Bioimpedance spectroscopy (BIS) is an easy tool to assess hydration status and body composition. However, its validity in athletes remains controversial. We investigated the validity of BIS on total body water (TBW) and body composition estimation in Japanese wrestlers and untrained subjects. TBW of 49 young Japanese male subjects (31 untrained, 18 wrestlers) were assessed using the deuterium dilution method (DDM) and BIS. De Lorenzo's and Moissl's equations were employed in BIS for TBW estimation. To evaluate body composition, Siri's 3-compartment model and published TBW/fat-free mass (FFM) ratio were applied in DDM and BIS, respectively. In untrained subjects, DDM and BIS with de Lorenzo's equation showed consistent TBW estimates, whereas BIS with Moissl's equation overestimated TBW ( < 0.001 vs. DDM). DDM and BIS with de Lorenzo's equation estimated FFM and percent of fat mass consistently, whereas BIS with Moissl's equation over-estimated and under-estimated them ( < 0.001 vs. DDM). In wrestlers, BIS with de Lorenzo's and Moissl's equations assessed TBW similarly with DDM. However, the Bland-Altman analysis revealed a proportional bias for TBW in BIS with de Lorenzo's equation