In vivo glycogen kinetics was estimated with the simultaneous use of indirect calorimetry and tracer technology in healthy humans during 24-h periods with low or moderate physical activity (1 or 3 exercise sessions each day). Two 13C-carbohydrates meals were administered at 9.30 a.m. and 1.30 p.m., and one 12C-carbohydrates meal at 6.30 p.m. Net carbohydrate oxidation (net CHO ox) was measured over a 24 h period by indirect calorimetry and oxidation of 13C-labelled carbohydrates (13C CHO ox) was estimated from 13CO2 production. Glycogen breakdown, assessed for the period 8.15 a.m.-6.30 p.m. as the difference between net CHO ox and 13C CHO ox, was increased 1.6 times with three exercise sessions [123.3 (SEM 8.0) g] versus one session [77.9 (SEM 7.7) g, P < 0.0001]. Carbohydrate balances over 24 h were close to zero under both conditions, indicating that glycogen breakdown was matched by an equivalent glycogen synthesis. It was concluded that simultaneous use of indirect calorimetry and tracer technology may make possible the estimation of glycogen kinetics in humans. Moderate physical activity enhanced both glycogen breakdown and synthesis. This stimulation of glycogen metabolism may therefore play a role in the enhanced insulin sensitivity induced by physical exercise.