Background <p>Human muscle reserve cells (MuRC) represent a quiescent MuSC population generated in vitro that exhibit heterogeneous Pax7 expression, with a Pax7^High subset in a deeper quiescent state. However, the conventional method of identifying Pax7^High cells involves intracellular staining, which limits their viability for functional studies. This work investigates whether autofluorescence (AF) could be used as a potential biomarker to identify functionally distinct human MuRC subpopulations.</p> Methods <p>Human myoblasts (MB) and MuRC were analysed for AF by fluorescence microscopy and flow cytometry. Cellular metabolic composition was assessed by NADH/NADPH quantification and lipid staining. Human MuRC subpopulations were sorted by AF intensity and analysed for Pax7 expression, cell cycle re-entry, proliferation, clonal expansion, and myogenic differentiation. In vivo transplantation of MuRC-AF^High and MuRC-AF^Low populations into immunodeficient mice assessed survival and regenerative potential using bioluminescence imaging and immunohistochemistry.</p> Results <p>Human MuRC exhibited a threefold increase in autofluorescence intensity compared to MB, with a peak at 405&#xa0;nm excitation, likely linked to a 1.6-fold increase in lipid content, while NADH/NADPH levels remained comparable. Flow cytometry identified MuRC-AF^High as a Pax7^High-enriched subpopulation, indicative of a deeper quiescent state. Functionally, MuRC-AF^High cells showed delayed cell cycle re-entry and slower proliferation yet maintained full differentiation capacity. In vivo, both MuRC-AF^High and MuRC-AF^Low survived transplantation, contributed to Pax7 + MuSC formation, and retained regenerative potential upon re-injury.</p> Conclusion <p>Autofluorescence enables the isolation of distinct human MuRC subpopulations. The AF^High subset contains a high proportion of Pax7^High cells and shows delayed activation yet retains engraftment efficiency that is comparable to that of the AF^Low subpopulation. These findings suggests that AF could be used as a biomarker to identify functionally distinct human muscle progenitor subsets while preserving their regenerative potential for future use.</p>