Background: Physical performance increased by controlled interventions of high-intensity intermittent training (HIIT); however, little is known about their influence as anti-aging and antioxidant effects, or their role in mitochondrial biogenesis. Purpose: This study aimed to determine the effects of HIIT for 12 weeks on melatonin function, lymphocyte cell apoptosis, oxidative stress on aging, and physical performance. Methods: Eighty healthy male subjects aged 18-65 years randomly participated in a HIIT-exercise training program for 12 weeks. Anthropometric analysis, cardiovascular fitness, total antioxidant capacity (TAC), lymphocyte count and apoptosis, and serum melatonin and cytochrome c oxidase (COX), were estimated for all subjects before and after HIIT-exercise training. HIIT training was performed in subjects for 12 weeks. Results: Data analysis showed a significant increase in the expression levels of the melatonin hormone (11.2 ± 2.3, p < 0.001), TAC (48.7 ± 7.1, p < 0.002), COX (3.7 ± 0.75, p < 0.001), and a higher percentage of lymphocyte apoptosis (5.2 ± 0.31, p < 0.003). In addition, there was an improvement in fitness scores (W; 196.5 ± 4.6, VO2max; 58.9 ± 2.5, p < 0.001), adiposity markers (p < 0.001); BMI, WHtR, and glycemic control parameters (p < 0.01); FG, HbA1c (%), FI, and serum C-peptide were significantly improved following HIIT intervention. Both melatonin and lymphocyte apoptosis significantly correlated with the studied parameters, especially TAC and COX. Furthermore, the correlation of lymphocyte apoptosis with longer exercise duration was significantly associated with increased serum melatonin following exercise training. This association supports the mechanistic role of melatonin in promoting lymphocyte apoptosis either via the extrinsic mediator pathway or via inhibition of lymphocyte division in the thymus and lymph nodes. Additionally, the correlation between melatonin, lymphocyte apoptosis, TAC, and COX activities significantly supports their role in enhancing physical performance. Conclusions: The main findings of this study were that HIIT exercise training for 12 weeks significantly improved adiposity markers, glycemic control parameters, and physical performance of sedentary older adult men. In addition, melatonin secretion, % of lymphocyte apoptosis, COX activities, and TAC as biological aging markers were significantly increased following HIIT exercise training interventions for 12 weeks. The use of HIIT exercise was effective in improving biological aging, which is adequate for supporting chronological age, especially regarding aging problems. However, subsequent studies are required with long-term follow-up to consider HIIT as a modulator for several cardiometabolic health problems in older individuals with obesity.