Background <p>Skeletal muscle is a central regulator of insulin sensitivity and glucose homeostasis. The ryanodine receptor 1 (RYR1) is highly expressed in skeletal muscle and plays a key role in myogenic differentiation. We hypothesize that RYR1⁺ extracellular vesicles (EVs) represent a skeletal muscle-derived EV subpopulation whose abundance and small RNA (smRNA) cargo are associated with aging, insulin action, and exercise responsiveness.</p> Methods <p>We tested this hypothesis through in vitro analyses of smRNAs of skeletal muscle-derived EVs and in vivo evaluation of their exercise responsiveness and association with insulin action in older adults. Using an integrated workflow combining centrifugation, polymer-based precipitation, and single-EV sorting, we isolated RYR1⁺ and RYR⁻ EVs secreted from myobundles—3D contractile skeletal muscle tissues engineered from primary muscle progenitor cells obtained from healthy donors (<i>n</i> = 6). We also isolated plasma EVs from 48 human participants and performed targeted high-resolution flow cytometry to evaluate EV biomarkers associated with aging, insulin action, and exercise responsiveness in older adults.</p> Results <p>By smRNA sequencing, compared to myobundle RYR⁻ EVs, RYR1⁺ EVs contained three unique microRNAs (miRNAs) and an additional 21 miRNAs with significantly greater abundance (including canonical myoMiRs miR-206, miR-1-3p, and miR-208a-3p). Of the 24 RYR1⁺ EV-enriched miRNAs, experimentally-supported mRNA targets (<i>n</i> = 422) are involved in pathways governing cell proliferation, apoptosis, senescence, insulin and glucose signaling. In two independent cohorts, including older adults with prediabetes or unknown prediabetes status, frequencies of RYR1⁺ EV subsets were significantly upregulated by chronic exercise with greater RYR1⁺ EV frequencies associated with better insulin action.</p> Conclusions <p>These complementary in vitro and in vivo data identify skeletal muscle-derived RYR1⁺ EVs as upregulated by exercise and as carriers of miRNAs linked to insulin action in older adults, including those with prediabetes. These results highlight skeletal muscle-derived EVs as novel biomarkers and potential mediators of systemic metabolic regulation and healthy aging.</p> Graphical Abstract <p></p>