Satellite cell contribution to unstressed diaphragm is higher compared to hind limb muscles, which is probably attributable to constant activation of this muscle to drive ventilation. Whether satellite cell depletion negatively impacts diaphragm quantitative and qualitative characteristics under stressed conditions in young and aged mice is unknown. We therefore challenged the diaphragm with prolonged running activity in the presence and absence of Pax7+ satellite cells in young and aged mice using an inducible Pax7^CreER‐R26R^DTA model. Mice were vehicle (Veh, satellite cell‐replete) or tamoxifen (Tam, satellite cell‐depleted) treated at 4 months of age and were then allowed to run voluntarily at 6 months (young) and 22 months (aged). Age‐matched, cage‐dwelling, Veh‐ and Tam‐treated mice without wheel access served as activity controls. Diaphragm muscles were analysed from young (8 months) and aged (24 months) mice. Satellite cell depletion did not alter diaphragm mean fibre cross‐sectional area, fibre type distribution or extracellular matrix content in young or aged mice, regardless of running activity. Resting in vivo diaphragm function was also unaffected by satellite cell depletion. Myonuclear density was maintained in young satellite cell‐depleted mice regardless of running, although it was modestly reduced in aged sedentary (–7%) and running (–19%) mice without satellite cells (P < 0.05). Using fluorescence in situ hybridization, we detected higher Pax3 mRNA+ cell density in both young and aged satellite cell‐depleted diaphragm muscle (P < 0.05), which may compensate for the loss of Pax7+ satellite cells.