In almost any sport, athletes undergoing dehydration often suffer from numerous dehydration related injuries. The purpose of this study was to evaluate the validity of acoustic method to detect changes in the hydration status of athletes after undergoing acute dehydration and a 2-hour rehydration protocol. The acoustic method of assessing body hydration status is based on the experimental fact that ultrasound speed in muscle is a linear function of the tissue water content. The assessment of water imbalance was conducted by measuring speed of ultrasound in the calf muscles using through transmission method. Eighty-two male and female collegiate athletes were examined to detect changes in hydration status before and after undergoing 3% acute dehydration. Results demonstrated that the changes of ultrasound velocity are in average about 1.1 m/s per 1% of body dehydration and ultrasound velocity in muscle potentially may serve as a measure of body hydration status. However, ultrasound speed measurement using through transmission mode implemented in this study is highly dependent on the positioning of the probe: even slight variation in the acoustic path results in significant changes in the measured values, which may results in unacceptable error. A solution to this problem is proposed and discussed. [NIH2R44AG042990.]