Both aging and physical activity can influence the amount of intramuscular connective tissue in skeletal muscle, but the impactof these upon specific extracellular matrix (ECM) proteins in skeletal muscle is unknown. We investigated the proteome profile ofintramuscular connective tissue by label-free proteomic analysis of cellular protein-depleted extracts from lateral gastrocnemiusmuscle of old (22–23 mo old) and middle-aged (11 mo old) male mice subjected to three different levels of regular physical activ-ity for 10 wk (high-resistance wheel running, low-resistance wheel running, or sedentary controls). We hypothesized that aging iscorrelated with an increased amount of connective tissue proteins in skeletal muscle and that regular physical activity can coun-teract these age-related changes. We found that dominating cellular proteins were diminished in the urea/thiourea extract, whichwas therefore used for proteomics. Proteomic analysis identified 482 proteins and showed enrichment for ECM proteins. Statisticalanalysis revealed that the abundances of 86 proteins changed with age. Twenty-three of these differentially abundant proteins wereidentified as structural ECM proteins (e.g., collagens and laminins) and all of these were significantly more abundant with aging. Nosignificant effect of training or interaction between training and advance in age was found for any proteins. Finally, we found a lowerprotein concentration in the urea/thiourea extracts from the old mice compared with the middle-aged mice. Ourfindings indicate thatthe intramuscular ECM solubility is affected by increased age but is not altered by physical training.NEW & NOTEWORTHYWe investigated the impact of aging and exercise on extracellular matrix components of intramuscularconnective tissue using proteomics. Middle-aged and old mice were subjected to three different levels of regular physical activ-ity for 10 wk (high-resistance wheel running, low-resistance wheel running, or sedentary controls). We prepared extracts ofextracellular matrix proteins depleted of cellular proteins. Ourfindings indicate that intramuscular connective tissue alters itssoluble protein content with age but is unaffected by training.