Purpose <p>Aging is associated with the loss of motoneurons and structural changes at the neuromuscular junction (NMJ), leading to functional decline in skeletal muscles. SEMA3A, a chemorepellent axonal guidance protein, may play a crucial role in these age-related changes. This study aimed to evaluate the effects of high-intensity interval training (HIIT) on the expression of SEMA3A and the pre- to postsynaptic coupling in the plantaris muscle of rats.</p> Methods <p>Twenty male rats (10 adult and 10 aged) were randomly assigned to one of four groups: adult control, adult trained, aged control, and aged trained. The exercise protocol involved treadmill training 5&#xa0;days a week for 6&#xa0;weeks. Immunohistochemical staining was used to assess synaptic SEMA3A protein levels and pre- to postsynaptic coupling. Real-time PCR was performed to quantify mRNA expression levels of SEMA3A, <i>Chrng</i>, <i>Chrnd</i>, and <i>Scn5a</i> (Nav1.5).</p> Results <p>Aging significantly increased the synaptic accumulation of SEMA3A protein (<i>P</i> = 0.001) and the expression of SEMA3A mRNA (<i>P</i> = 0.001), as well as other denervation-related genes in the plantaris muscle. HIIT led to a notable decrease in synaptic SEMA3A protein and gene expression in both adult and aged rats (<i>P</i> &lt; 0.05), compared to their respective control groups. Pre- to postsynaptic coupling decreased with aging, but HIIT significantly improved this coupling, particularly in the adult trained group (<i>P</i> &lt; 0.05). In aged rats, HIIT attenuated the age-related decline in pre- to postsynaptic coupling and reduced the expression of genes linked to denervation.</p> Conclusion <p>The results suggest that HIIT may help mitigate age-related denervation and muscle atrophy by downregulating SEMA3A expression and improving NMJ integrity. These findings highlight the potential of high-intensity exercise to preserve neuromuscular health and slow the progression of age-related muscle deterioration. </p>