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We hypothesized that the reduction of O2-carrying capacity caused by the withdrawal of ∼450 ml blood would result in slower phase II O2 uptake V̇O2 kinetics, a lower V̇O2peak and a reduced time to exhaustion during severe-intensity cycle exercise. Eleven healthy subjects (mean ±S.D. age 23 ± 6 years, body mass 77.2 ± 11.0 kg) completed ‘step’ exercise tests from unloaded cycling to a severe-intensity work rate (80% of the difference between the predetermined gas exchange threshold and the V̇O2peak) on two occasions before, and 24 h following, the voluntary donation of ∼450 ml blood. Oxygen uptake was measured breath-by-breath, and V̇O2 kinetics estimated using non-linear regression techniques. The blood withdrawal resulted in a significant reduction in haemoglobin concentration (pre: 15.4 ± 0.9 versus post: 14.7 ± 1.3 g dl−1; 95% confidence limits (CL): −0.04, −1.38) and haematocrit (pre: 44 ± 2 versus post: 41 ± 3%; 95% CL: −1.3, −5.1). Compared to the control condition, blood withdrawal resulted in significant reductions in V̇O2peak (pre: 3.79 ± 0.64 versus post: 3.64 ± 0.61 l min−1; 95% CL: −0.04, − 0.27) and time to exhaustion (pre: 375 ± 129 versus post: 321 ± 99 s; 95% CL: −24, −85). However, the kinetic parameters of the fundamental V̇O2 response, including the phase II time constant (pre: 29 ± 8 versus post: 30 ± 6 s; 95% CL: 5, −3), were not altered by blood withdrawal. The magnitude of the V̇O2 slow component was significantly reduced following blood donation owing to the lower V̇O2peak attained. We conclude that a reduction in blood O2-carrying capacity, achieved through the withdrawal of ∼450 ml blood, results in a significant reduction in V̇O2peak and exercise tolerance but has no effect on the fundamental phase of the V̇O2 on-kinetics during severe-intensity exercise.