Abstarct: Introduction and Objective: Understanding the immediate physiological responses to exercise is essential for designing safe training programs; however, the response of muscle damage markers to a single session of high-intensity interval training (HIIT) in inactive overweight men remains unclear. The aim of the present study was to examine the acute response of muscle damage markers to a single session of high-intensity interval training in inactive overweight men. Methods: The present study was a quasi-experimental study with a pretest–posttest control group design, conducted to investigate the acute response of muscle damage markers to a single session of high-intensity interval training (HIIT) in inactive overweight men. The study population consisted of inactive overweight men aged 30–40 years with a body mass index (BMI) of 25–29.9 kg/m² in Shahroud, Iran. A total of 20 participants were selected and assigned to two groups: the high-intensity interval training group (n = 10) and the control group (n = 10). Following a familiarization session, participants underwent pretest blood sampling at 8:00 a.m. after 20 minutes of rest under controlled conditions. The experimental group then performed a single HIIT session consisting of a 5-minute warm-up at 50–60% of maximum heart rate (HRmax), eight 1-minute bouts of exercise at 85–90% of HRmax, each separated by 1 minute of active recovery at 30–40% of HRmax, followed by a 5-minute cooldown at 40–50% of HRmax. The control group remained at rest for the same duration. Posttest blood samples were collected immediately after completion of the exercise or rest period. Serum creatine kinase (CK) and lactate dehydrogenase (LDH) concentrations were measured using the enzyme-lixnked immunosorbent assay (ELISA) method. Data were analyzed using descxriptive statistics and a 2 × 2 mixed-design repeated-measures analysis of variance (ANOVA). The assumptions of normality and homogeneity of variance were assessed using the Shapiro–Wilk and Levene’s tests, respectively, and Box’s M test was used to assess the homogeneity of covariance matrices. Pairwise comparisons were performed with Bonferroni adjustment. The significance level was set at 0.05. Results: The time × group interaction effect was significant for both muscle damage markers (P < 0.001). Creatine kinase in the HIIT group increased from 193.66 ± 41.23 to 216.58 ± 40.00 U/L, whereas the control group showed no notable change (from 194.41 ± 32.64 to 193.76 ± 34.39 U/L) (P < 0.001, partial η² = 0.770). Lactate dehydrogenase also increased in the training group from 249.37 ± 30.53 to 275.32 ± 38.00 U/L, while the control group showed only a slight change (from 255.66 ± 28.12 to 251.56 ± 28.50 U/L) (P < 0.001, partial η² = 0.717). Conclusion: A single session of high-intensity interval training significantly increased serum levels of creatine kinase and lactate dehydrogenase in inactive overweight men. This increase reflects an acute muscle damage response and a transient increase in muscle cell membrane permeability due to the mechanical and mextabolic stress induced by HIIT. Therefore, despite the effectiveness of HIIT as a training stimulus, safety considerations and adequate recovery time appear necessary in the initial training programs for this population.