Purpose: Changes in biophysiological markers related to perceived burnout and self-comfort were tested in this study. Method: Forty-two student-athletes from middle and high school grades were evaluated for burnout, salivary cortisol levels, measures of arousal-related physiological markers (i.e., blood volume pulse; BVP), galvanic skin response (GSR), and respiratory rate, and self-comfort variables during the last two weeks of the season. Using self-comfort theory as its conceptual framework, we examined burnout through a conceptual model in which feeling of discomfort with the self was related to biophysiological markers affecting perceived burnout. The proposed model was tested by using a partial least squares structural equation modeling (PLS-SEM). Results: The main findings indicate that increased self-discomfort is significantly (p < 0.001) associated with increased salivary cortisol ([beta] = - 0.189) along with a significant (p = 0.050) decrease in GSR ([beta] = - 0.259). Increased salivary cortisol is significantly (p < 0.001) associated with increased burnout ([beta] = 0.242). Conclusion: The findings partially support the model and encourage further effort to capture the burnout syndrome through the integration of biological and psychological markers.