Mechanical Gas Exchange Simulation Systems (GESS) have never been used to compare different breath-by-breath oxygen uptake calculation algorithms. Oxygen uptakes were calculated for each GESS cycle by the "Expiration-only" algorithm (estimating inspiratory volume from the expiratory one), and by two "alveolar" algorithms (both processing inspiratory and expiratory flows and designed to account for the changes in lung gas stores). The volume of oxygen stored in the GESS from one cycle to the subsequent one was either maintained constant or increased/decreased by changing the pumped gas volumes. Overlapping oxygen uptakes were obtained maintaining constant the volume of oxygen stored (grand average: 0.420 ± 0.019 L/min, p = ns). The "Expiration-only" algorithm overestimated the decreases of the stored oxygen by 34%, whereas the "alveolar" algorithms underestimated the increases by 25%; in the other conditions, the changes of the stored oxygen were appropriately accounted for. The use of "alveolar" algorithms is recommended, particularly so when abrupt changes in the stored oxygen volume occur.