It is generally thought that intracellular pH (pHi) of skeletal muscle falls at least 0.5 units during intense activity, but evidence on natural (i.e., voluntary, two-legged (2L)) locomotor activity in man has exclusively come from invasive studies of upper leg muscle. Here, noninvasive (31)P nuclear magnetic resonance spectroscopy ((31)P NMRS) was used to study human quadriceps muscle energetics and pHi during incremental bicycling exercise to exhaustion in six normally active subjects. Cellular energy charge (CEC; [PCr]/([PCr]+[Pi])) linearly (r 0.90) dropped 83 +/- 3% during ramp exercise to exhaustion from 0.92 +/- 0.01 at rest to 0.16 +/- 0.03 at maximal sustained work rate (WR) (166+/-17 W; range: 108-223 W). Surprisingly, pHi likewise dropped linearly (r 0.82) no more than 0.2 units over the entire range of WR between rest and maximal (pHi 7.08+/-0.01 and 6.84+/-0.02, respectively). But after termination of exercise pHi dropped rapidly to textbook acidic values of 6.6 explaining classic biopsy results. Comparative coresponse analysis of pHi and CEC changes during 2L- vs. 1L-cycling showed that homeostatic control of quadriceps pHi during bicycling is robust and unique to natural locomotor exercise. These results highlight the robustness of the integrative set of physicochemical and physiological control mechanisms in acid-base balance during natural locomotor activity in man.