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The aim of this study was to determine how ATP synthesis and contractility in vivo are altered by ischaemia in working human skeletal muscle. The hypotheses were: (1) glycolytic flux would be higher during ischaemic (ISC) compared to free‐flow (FF) muscle contractions, in compensation for reduced oxidative ATP synthesis, and (2) ischaemic muscle fatigue would be related to the accumulation of inhibitory metabolic by‐products rather than to the phosphorylation potential ([ATP]/[ADP][P_i]) of the muscle. Twelve healthy adults (6 men, 6 women) performed six intermittent maximal isometric contractions of the ankle dorsiflexors (12 s contract, 12 s relax), once with intact blood flow and once with local ischaemia by thigh cuff inflation to 220 Torr. Intracellular phosphorous metabolites and pH were measured non‐invasively with magnetic resonance spectroscopy, and rates of ATP synthesis through oxidative …