To date, no physical response data are available for one of the most popular eSport games, Overwatch. The purpose of this investigation was to describe the stress signaling associated with competitive Overwatch play and to understand how acute hormonal responses may affect performance. Thirty-two male college-aged gamers (age: 21.3 6 2.7 years; estimated time played per week: 18 6 15 hours) completed the study. Subjects were randomly assigned to a 6-player team to compete in a tournament-style match. Salivary measures of cortisol and testosterone were collected immediately before (PRE) and after (POST) the first-round game, with the heart rate recorded continuously during the match. The mean characteristics were calculated for each variable and comparisons made by the skill level. Significance was defined as p # 0.05. There were no differences in measures of salivary cortisol. A differential response pattern was observed by the skill level for testosterone. The low skill group displayed a significant increase in testosterone with game play (mean 6 SD, testosterone PRE: 418.3 6 89.5 pmol•L 21 , POST: 527.6 6 132.4 pmol•L 21 , p , 0.001), whereas no change was observed in the high skill group. There were no differences in heart rate characteristics between skill groups. Overall, the average heart rate was 107.2 6 17.8 bpm with an average max heart rate of 133.3 6 19.1 bpm. This study provides unique physiological evidence that a sedentary Overwatch match modulates endocrine and cardiovascular responses, with the skill level emerging as a potential modulator.