The onset of intracellular acidosis during muscular exercise has been generally attributed to an activation or hyperactivation of non oxidative ATP production but has never been analysed quantitatively in terms of H⁺ balance i.e. production/removal mechanisms. In an attempt to address this issue, we have analysed the relationship between intracellular acidosis and H⁺ balance during exercise bouts in seven healthy volunteers. Each subject performed a 6-minute ramp rythmic exercise (finger flexions) at low-frequency (LF, 0.47 Hz) leading to slight acidosis and at high frequency (HF, 0.85 Hz) inducing a larger acidosis. Metabolic changes were recorded using ³¹P MR spectroscopy . The onset of intracellular acidosis was statistically identified after 3 and 4 minutes of exercise for HF and LF protocols, respectively. A detailed investigation of H⁺ balance indicated that for both protocols non oxidative ATP production occurred before pH changed. Both at the low and high frequency protocols, H⁺ consumption through the creatine kinase equilibrium was constant in the face of increasing proton generation and efflux. For both protocols, changes in pH were not recorded as long as sources and sinks for H⁺ approximately balanced. In contrast, a significant acidosis occurred after 4 minutes of LF exercise and 3 minutes of HF exercise while the rise in H⁺ generation exceeded the rise in H⁺ efflux at a nearly constant H⁺ uptake associated with PCr breakdown. We clearly demonstrated in the present study that intracellular acidosis recorded in exercising muscle does not occur exclusively as a result of non oxidative ATP production, but rather reflects changes in overall H⁺ balance.