The flow response time theory allows the global assessment of a metabolic pathway. This study describes the first application of this concept to explore glycolysis on human skeletal muscle extracts. The muscle extract is used to convert glucose or glucose-6-phosphate into glycerol-phosphate through the first part of glycolysis. The functioning of the experimental model is assayed by a continuous recording of the reduced nicotinamide adenine dinucleotide decay in a spectrophotometer. This measurement method was applied to normal and pathologic human skeletal muscles. The aerobic (J(A)) and anaerobic (J(B)) fluxes and the time (t99) needed for the transition from J(A) to J(B) were measured under a wide clinical temperature range (30 degrees C to 40 degrees C). The two studied muscle types (gluteus maximus and tibialis anterior) have similar glycolytic flux values, with an identical functional modality. The thermal response of glycolysis is not linear, with a high thermal sensitivity in the hypothermic range (30 degrees C to 38 degrees C) and a thermal insensitivity in the hyperthermic range (37 degrees C to 40 degrees C). On the same type of muscle (tibialis anterior), a pathologic process can induce different variations in the glycolysis patterns, but further data are needed to clear this point. The flow response time concept allows an accurate assessment of glycolysis in the human skeletal muscle, whether normal or pathologic. This approach is interesting for evaluating the global influence of different stimulations on a metabolic pathway, such as temperature variation.