Abstarct: Introduction and Purpose: Overweight and obesity may be associated with alterations in the hormonal balance of the hypothalamic–pituitary–adrenal and hypothalamic–pituitary–gonadal axes and can affect the hormonal response to intense exercise. High-intensity interval training (HIIT) has been considered as an efficient and time-effective intervention; however, the acute response of cortisol, testosterone, and the testosterone-to-cortisol ratio to a single session of HIIT in inactive overweight men is not yet fully understood, and the available evidence is limited and somewhat inconsistent. The purpose of the present study was to investigate the acute response of cortisol, testosterone, and the testosterone-to-cortisol ratio to a single session of high-intensity interval training in inactive overweight men. Methods: The present study was a semi-experimental design with a pretest–posttest and a control group. The statistical population consisted of inactive overweight men aged 30 to 40 years with a body mass index of 25 to 29.9 kg/m² in Shahroud. Twenty participants were selected as the study sample and assigned to two groups: high-intensity interval training (10 participants) and control (10 participants). After a familiarization session, participants underwent pretest blood sampling at 8:00 AM after 20 minutes of rest under controlled conditions. The experimental group then performed a single session of high-intensity interval training consisting of 5 minutes of warm-up at 50–60% of maximum heart rate, 8 one-minute bouts of activity at 85–90% of maximum heart rate, with one minute of active recovery at 30–40% of maximum heart rate between bouts, and 5 minutes of cool-down at 40–50% of maximum heart rate. The control group remained at rest for the same duration. Immediately after the completion of exercise or the rest period, posttest blood sampling was performed. Serum concentrations of cortisol and testosterone were measured using the ELISA method. Data were analyzed using descxriptive statistics and repeated-measures analysis of variance in a 2×2 mixed design. Statistical assumptions were checked using the Shapiro–Wilk, Levene, and Box's M tests, and pairwise comparisons were performed with Bonferroni correction. A significance level of 0.05 was considered. Results: The time × group interaction was significant for cortisol (F = 5.552, p = 0.030, partial η² = 0.236), testosterone (F = 5.080, p = 0.037, partial η² = 0.220), and the testosterone-to-cortisol ratio (F = 4.758, p = 0.043, partial η² = 0.209). Cortisol in the high-intensity interval training group increased from 143.95 ± 20.88 to 161.85 ± 25.73 ng/mL, while the control group showed a smaller change (from 158.20 ± 27.20 to 161.00 ± 30.15). Testosterone in the high-intensity interval training group increased from 5.87 ± 0.36 to 6.31 ± 0.35 ng/mL, whereas the control group showed a smaller change (from 5.61 ± 0.56 to 5.81 ± 0.62). The testosterone-to-cortisol ratio in the high-intensity interval training group decreased from 0.0416 ± 0.0069 to 0.0397 ± 0.0056, while the control group showed a slight increase (from 0.0364 ± 0.0069 to 0.0375 ± 0.0094). Conclusion: Conclusion: A single session of high-intensity interval training in inactive overweight men significantly increased serum cortisol and testosterone and significantly decreased the testosterone-to-cortisol ratio. These findings indicate activation of the hypothalamic–pituitary–adrenal axis and the hypothalamic–pituitary–gonadal axis and the induction of a transient catabolic state in acute response to this type of exercise. Given this response, adequate recovery periods and gradual progression of exercise intensity appear necessary in this population.