Middle-aged (37Y) and older master (50Y) highly-trained athletes performed a 24-h treadmill run (24TR). Vastus lateralis muscle biopsies were collected before and after the run and assessed for proteomics, fiber morphometry, intramyocellular lipid droplets (LD), mitochondrial oxidative activity, extracellular matrix (ECM) and micro-vascularisation. Before 24TR, muscle fiber type morphometry, intramyocellular LD, oxidative activity, ECM and micro-vascularisation were similar between master and middle-aged runners. For 37Y runners, 24TR was associated with ECM thickening, increased capillary tortuosity, and 89% depletion of LD in type-I fibers. In contrast for 50Y runners, 24TR did not alter ECM and capillarization and poorly depleted LDs. Moreover, impaired SDH activity and functional class scoring of proteomes suggested reduced oxidative phosphorylation exclusively in 50Y muscle post-24TR. Collectively, our data support that middle-aged and master endurance athletes exhibit distinct transient plasticity in response to a single bout of ultra-endurance exercise, which may constitute early signs of muscle aging for master athletes.