Purpose The role of splenic emptying in O 2 transport during aerobic exercise still remains a matter of debate. Our study compared the differences in spleen volume changes between aerobically trained and untrained individuals during step-transition supine cycling exercise at moderate-intensity. We also examined the relationship between spleen volume changes, erythrocyte release, and O 2 uptake parameters. Methods Fourteen healthy men completed all study procedures, including a detailed medical examination, supine maximal O 2 uptake ( V ˙ O 2 max.) test, and three step-transitions from 20 W to a moderate-intensity power output, equivalent to V ˙ O 2 uptake at 90% gas exchange threshold. During these step-transitions pulmonary V ˙ O 2 p , near-infrared spectroscopy of the vastus lateralis , and cardiovascular responses were continuously measured. In parallel, minute-by-minute ultrasonic measurements of the spleen were performed. Blood samples were taken before and immediately after step-transition cycling. Results On average, V ˙ O 2 max. was 10 mL kg min −1 ( p  = 0.001) higher in trained compared to their aerobically untrained peers. In response to supine step-transition cycling, the splenic volume was significantly reduced, and the largest reduction (~ 106 to 115 mL, ~ 38%, p  = 0.001) was similar in both aerobically trained and untrained individuals. Erythrocyte concentration and platelet count transiently increased after exercise cessation, with no differences observed between groups. However, the vastus lateralis deoxygenation amplitude was 30% ( p  = 0.001) greater in trained compared to untrained individuals. No associations existed between: (i) spleen volumes at rest (ii) spleen volume changes (%), (iii) resting hematocrit and oxygen uptake parameters. Conclusion Greater splenic emptying and subsequent erythrocyte release do not lead to a slower τ V ˙ O 2 p , regardless of individual V ˙ O 2 max. readings.