The sirtuins (SIRT) protein family and the mechanistic/mammalian target of rapamycin (mTOR) are intracellular molecules that have been involved in the regulation of several biological processes, as well as in various aging-related processes. This pilot study, in small scale, aimed to analyze the effects of an 8-week physical exercise program on SIRT3 and mTOR levels in lymphocytes, as well as on lipid peroxidation in middle aged and older men. A total of 9 participants aged between 56 and 73 years were enrolled in an 8-week physical exercise program comprising cardiovascular and high-intensity interval training. The program involved three sessions per week, each lasting 45–60 min, conducted on non-consecutive days. Tests were conducted before and after the experimental period (pre- and post-training). Assessments included a vertical jump, 20 m velocity, ball throwing, and an aerobic capacity test. Lipid peroxidation (MDA) was measured in plasma as an oxidative stress biomarker. Additionally, sirtuin 3 (SIRT3/β-actin) and mTOR (mTOR/β-actin) levels were measured in isolated lymphocytes extracted from venous blood. Following the exercise training period, our results demonstrated a significant improvement in aerobic capacity (pre-training: 615.4 ± 45.3 m; post-training: 687.2 ± 34.6 m; t = −2.521; p = 0.012) and 20 m velocity (pre-training: 4.6 ± 0.5 s; post-training: 4.3 ± 0.3 s; t = −2.023; p = 0.04). Concerning blood variables, there was a significant decrease in mTOR levels (pre-training: 0.857 ± 0.593; post-training: 0.214 ± 0.097; t = −2.547; p = 0.011), while no changes were observed in SIRT3 (pre-training: 0.608 ± 0.404; post-training: 0.516 ± 0.390; t = 0.533; p = 0.594) and MDA (pre-training: 8420 ± 4615; post-training: 8800 ± 3163; t = −0.533; p = 0.594). The notable reduction in mTOR levels in lymphocytes following the 8-week physical exercise program suggests a potential role of exercise in modulating immune cell dynamics, particularly in middle-aged and older individuals. Furthermore, the exercise regimen resulted in improvements in physical function, including enhanced aerobic capacity and walking velocity.