We recently reported lower glycolytic flux (ATPGLY) and increased reliance on oxidative ATP synthesis (ATPOX) in contracting muscle of older compared to young humans. To further investigate this age-related difference in the pathways of ATP synthesis, we used magnetic resonance spectroscopy to determine the rates of ATPOX, ATPGLY and net phosphocreatine hydrolysis in vivo during maximal muscle contractions under free-flow (FF) and ischaemic (ISC) conditions in the ankle dorsiflexors of 20 young (27 ± 3 years; 10 male, 10 female) and 18 older (70 ± 5 years; 10 male, 8 female) adults. We hypothesized that ATPGLY would be higher in young compared to old during FF contractions, but that old would be unable to increase ATPGLY during ISC to match that of the young, which would suggest impaired glycolytic ATP synthesis with old age. Peak glycolytic flux during FF was lower in older (0.8 ± 0.1 mm ATP s−1) compared to young (1.4 ± 0.1 mm ATP s−1, P < 0.001) subjects. During ISC, peak ATPGLY increased in old to a level similar to that of young (1.4 ± 0.2 mm ATP s−1, 1.3 ± 0.2 mm ATP s−1, respectively; P= 0.86), suggesting that glycolytic function remains intact in aged muscle in vivo. Notably, older adults fatigued less than young during both FF and ISC (P≤ 0.004). These results provide novel evidence of unimpaired in vivo glycolytic function in the skeletal muscle of older adults during maximal isometric dorsiflexion, and suggest a potential role for differences in metabolic economy and as a result, metabolite accumulation, in the fatigue resistance of the old.