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 phosphorylation, anaerobic glycolysis, and the creatine kinase reaction were determined. The force-time integral declined more during ISC (66 +/- 3% initial) than FF (75 +/- 2% initial, P = 0.002), indicating greater fatigue in ISC. [ATP] was preserved in both protocols, indicating matching of ATP production and use under both conditions. Glycolytic flux (mm s(-1)) was similar during FF and ISC (P = 0.16). Total ATP synthesis rate was lower during ISC, despite adjustment for the greater muscle fatigue in this condition (P < 0.001). Fatigue was linearly associated with diprotonated inorganic phosphate (FF r = 0.94 +/- 0.01, ISC r = 0.92 +/- 0.02), but not phosphorylation potential. These data provide novel evidence that ATP supply and demand in vivo are balanced in human skeletal muscle during ischaemic work, not through higher glycolytic flux, but rather through increased metabolic economy and decreased rates of ATP consumption as fatigue ensues.