Both aging and physical activity can influence the amount of intramuscular connective tissue in skeletal muscle, but the impact of these upon specific extracellular matrix (ECM) proteins in skeletal muscle is unknown. We investigated the proteome profile of intramuscular connective tissue by label-free proteomic analysis of cellular protein-depleted extracts from lateral gastrocnemius muscle of old (22-23 mo old) and middle-aged (11 mo old) male mice subjected to three different levels of regular physical activity for 10 wk (high-resistance wheel running, low-resistance wheel running, or sedentary controls). We hypothesized that aging is correlated with an increased amount of connective tissue proteins in skeletal muscle and that regular physical activity can counteract these age-related changes. We found that dominating cellular proteins were diminished in the urea/thiourea extract, which was therefore used for proteomics. Proteomic analysis identified 482 proteins and showed enrichment for ECM proteins. Statistical analysis revealed that the abundances of 86 proteins changed with age. Twenty-three of these differentially abundant proteins were identified as structural ECM proteins (e.g., collagens and laminins) and all of these were significantly more abundant with aging. No significant effect of training or interaction between training and advance in age was found for any proteins. Finally, we found a lower protein concentration in the urea/thiourea extracts from the old mice compared with the middle-aged mice. Our findings indicate that the intramuscular ECM solubility is affected by increased age but is not altered by physical training.NEW & NOTEWORTHY We investigated the impact of aging and exercise on extracellular matrix components of intramuscular connective tissue using proteomics. Middle-aged and old mice were subjected to three different levels of regular physical activity for 10 wk (high-resistance wheel running, low-resistance wheel running, or sedentary controls). We prepared extracts of extracellular matrix proteins depleted of cellular proteins. Our findings indicate that intramuscular connective tissue alters its soluble protein content with age but is unaffected by training.