The oObjective of the study was to investigate whether the occurrence of cardiac–locomotor synchronization (CLS) affects oxygen pulse (O₂ pulse, mL/beat) during walking. Twelve healthy men were studied under two treadmill protocols. The CLS protocol involved subjects walking at a frequency of their heart rate (HR) to induce CLS. The free protocol (reference) involved subjects walking at a self-selected cadence. The treadmill load was equal between the two protocols and was adjusted so that the subject’s HR was maintained at approximately 120 bpm. Electrocardiographic signals, foot switch signals, and oxygen consumption (VO₂) were measured continuously for 10 min after the heart rate reached a steady state. VO₂, O₂ pulse, and mean HR were calculated. VO₂ and O₂ pulse were significantly higher in subjects in the CLS protocol compared to those in the free protocol. However, mean HR was not different between the two groups. The synchronization strength was significantly related to the increase in O₂ pulse in subjects in the CLS protocol compared with those in the free protocol. These results suggest that the occurrence of CLS enhances O₂ pulse by increasing the strength of CLS during walking.