This study investigated the associations among age, physical activity level, knee muscle function, and neuromuscular junction (NMJ) biomarkers in a cohort of 80 middle-aged and young men residing in the Republic of Korea. Despite comparable levels of physical activity between the groups, the middle-aged participants exhibited significantly higher body fat percentage, elevated levels of the neurodegeneration marker neurofilament light chain (NfL), and a marked decline in lower-extremity muscle function compared to their younger counterparts. Advancing age was negatively associated with knee extensor peak torque, body weight–normalized torque (BW/PT), and the rate of torque development at 0.18 s (RTD@0.18s). In contrast, higher physical activity levels were positively correlated with certain indicators of muscle function and were associated with lower circulating concentrations of the NMJ degeneration marker, C-terminal agrin fragment (CAF). These findings suggest that neuromuscular decline and muscle function deterioration may begin as early as middle age. The results underscore the importance of implementing tailored exercise regimens and lifestyle interventions to preserve neuromuscular health and prevent early-onset muscle loss.