The longstanding problem of rapid inactivation of the glycolytic pathway in skeletal muscle after contraction was investigated using 31P NMR spectroscopy and computational modeling. Accumulation of phosphorylated glycolytic intermediates (hexose-mono-phosphates) during cyclic contraction and subsequent turnover during metabolic recovery was measured in vivo in human quadriceps muscle using dynamic 31P NMR spectroscopy. Hexose-mono-phosphates concentration in muscle peaked 40s into metabolic recovery from maximal contractile work at 6.9+1.3 mM (mean+SD; n=8) and subsequently declined at a rate of 0.009+0.001 mM/s. It was next tested if current knowledge of the kinetic controls in the glycolytic pathway in muscle integrated in the Lambeth and Kushmerick computational model of skeletal muscle glycolysis, explained the experimental data. It was found that the model underestimated the magnitude of deactivation of the glycolytic pathway in resting muscle resulting in depletion of glycolytic intermediates and substrate for oxidative ATP synthesis. Numerical analysis of the model identified phosphofructokinase and pyruvate-kinase as the kinetic control sites involved in deactivation of the glycolytic pathway. Ancillary 100-fold inhibition of both phosphofructokinase and pyruvate-kinase was found necessary to correctly predict glycolytic intermediate and ADP concentrations in resting human muscle. Incorporation of this information in the model resulted in highly improved agreement between predicted and measured in vivo hexose-mono-phosphates dynamics in muscle following contraction. We concluded that silencing of the glycolytic pathway in muscle following contraction is most likely mediated by phosphofructokinase and pyruvate-kinase inactivation on a timescale of seconds and minutes, respectively, and necessary to prevent depletion of vital cellular substrates.