Sestrins and autophagy deficiencies are associated with several aging-related organic dysfunctions and metabolic disorders. Here we evaluate the effects of acute exercise on Sestrin 2 (Sesn2) protein content and autophagy markers in the skeletal muscle of experimental models of aging. Twenty-four months-old C57BL/6J male mice were submitted to a single bout of swimming exercise and the gastrocnemius muscle was evaluated by Western blot. Transcriptomic and phenotypic analysis were also performed by using strains of genetically-diverse BXD mice. The bioinformatics analysis showed a negative correlation between Sesn2 mRNA levels in the skeletal muscle and body weight gain, plasma triglycerides and fasting glucose and positive correlation with several autophagic markers in the muscle of BXD mice. Consistent with these findings, low levels of Sesn2 protein content were observed in the gastrocnemius muscle of C57BL/6J old mice when compared to young group. Interestingly, the acute aerobic exercise induced Sesn2 accumulation and modulated several markers of autophagy in the gastrocnemius muscle old mice, including unc-51-like kinase-1 (Ulk1) phosphorylation and the protein levels of Atg5, Atg7, p62 and LC3-II. Finally, exercise increased insulin sensitivity in old animals, as demonstrated by kITT. Taken together, these findings demonstrated the acutely, aerobic physical exercise recovers Sestrin 2 protein content and induces autophagy in the skeletal muscle of old mice, contributing with the improvement of insulin sensitivity an aging animal model. •Downregulation of Sestrin2 in the skeletal muscle of old mice.•Exercise stimulates Sestrin2 accumulation in the skeletal muscle of old mice.•Physical exercise elicits autophagy in the skeletal muscle of old mice.