Purpose The endurance training (ET)-induced increases in peak oxygen uptake (V O 2peak) and cardiac output (Q peak) during upright cycling are reversed to pre-ET levels after removing the training-induced increase in blood volume (BV). We hypothesised that ET-induced improvements in V O 2peak and Q peak are preserved following phlebotomy of the BV gained with ET during supine but not during upright cycling. Arteriovenous O 2 difference (a-vO 2 diff; V O 2 /Q), cardiac dimensions and muscle morphology were studied to assess their role for the V O 2peak improvement. Methods Twelve untrained subjects (V O 2peak : 44 ± 6 ml kg −1 min −1) completed 10 weeks of supervised ET (3 sessions/ week). Echocardiography, muscle biopsies, haemoglobin mass (Hb mass) and BV were assessed pre-and post-ET. V O 2peak and Q peak during upright and supine cycling were measured pre-ET, post-ET and immediately after Hb mass was reversed to the individual pre-ET level by phlebotomy. Results ET increased the Hb mass (3.3 ± 2.9%; P = 0.005), BV (3.7 ± 5.6%; P = 0.044) and V O 2peak during upright and supine cycling (11 ± 6% and 10 ± 8%, respectively; P ≤ 0.003). After phlebotomy, improvements in V O 2peak compared with pre-ET were preserved in both postures (11 ± 4% and 11 ± 9%; P ≤ 0.005), as was Q peak (9 ± 14% and 9 ± 10%; P ≤ 0.081). The increased Q peak and a-vO 2 diff accounted for 70% and 30% of the V O 2peak improvements, respectively. Markers of mitochondrial density (CS and COX-IV; P ≤ 0.007) and left ventricular mass (P = 0.027) increased. Conclusion The ET-induced increase in V O 2peak was preserved despite removing the increases in Hb mass and BV by phlebotomy, independent of posture. V O 2peak increased primarily through elevated Q peak but also through a widened a-vO 2 diff, potentially mediated by cardiac remodelling and mitochondrial biogenesis. Keywords Blood volume • Cardiac output • Echocardiography • Haemoglobin mass • Maximal oxygen uptake • Peripheral adaptations • Supine cycling Abbreviations a-vO 2 diff Arteriovenous oxygen difference BV Blood volume CO Carbon monoxide COX-IV Cytochrome c oxidase subunit 4 CS Citrate synthase EDV End-diastolic volume ESV End-systolic volume ET Endurance training HAD Hydroxyacyl-CoA dehydrogenase [Hb] Haemoglobin concentration Hb mass Haemoglobin mass HR Heart rate HR peak Peak heart rate [La] Blood lactate concentration LV Left ventricle MCHC Mean corpuscular haemoglobin concentration MTT Mean transit time PV Plasma volumė Q Cardiac outpuṫ Q peak Peak cardiac output Communicated by Peter Krustrup.

When exercising with a small muscle mass, the mass‐specific O2 delivery exceeds the muscle oxidative capacity resulting in a lower O2 extraction compared with whole‐body exercise. We elevated the muscle oxidative capacity and tested its impact on O2 extraction during small muscle mass exercise. Nine individuals conducted six weeks of one‐legged knee extension (1L‐KE) endurance training. After training, the trained leg (TL) displayed 45% higher citrate synthase and COX‐IV protein content in vastus lateralis and 15%‐22% higher pulmonary oxygen uptake ( trueV˙normalO2peak) and peak power output ( normalW˙peak) during 1L‐KE than the control leg (CON; all P < .05). Leg O2 extraction (catheters) and blood flow (ultrasound Doppler) were measured while both legs exercised simultaneously during 2L‐KE at the same submaximal power outputs (real‐time feedback‐controlled). TL displayed higher O2 extraction than CON (main effect: 1.7 ± 1.6% points; P = .010; 40%‐83% of normalW˙peak) with the largest between‐leg difference at 83% of normalW˙peak (O2 extraction: 3.2 ± 2.2% points; arteriovenous O2 difference: 7.1 ± 4.8 mL· L−1; P < .001). At 83% of normalW˙peak, muscle O2 conductance (DMO2; Fick law of diffusion) and the equilibration index Y were higher in TL (P < .01), indicating reduced diffusion limitations. The between‐leg difference in O2 extraction correlated with the between‐leg ratio of citrate synthase and COX‐IV (r = .72‐.73; P = .03), but not with the difference in the capillary‐to‐fiber ratio (P = .965). In conclusion, endurance training improves O2 extraction during small muscle mass exercise by elevating the muscle oxidative capacity and the recruitment of DMO2, especially evident during high‐intensity exercise exploiting a larger fraction of the muscle oxidative capacity.