Regional anaesthesia to attenuate skeletal muscle afferent feedback abolishes the exercise-induced increase in middle cerebral artery mean blood velocity (MCA Vmean). However, such exercise-related increases in cerebral perfusion are not preserved during post exercise muscle ischaemia (PEMI) where the activation of metabolically sensitive muscle afferents is isolated. We tested the hypothesis that a hyperventilation-mediated decrease in the arterial partial pressure of CO2, hence cerebral vasoconstriction, masks the influence of muscle metaboreceptor stimulation on MCA Vmean during PEMI. Ten healthy men (20 ± 1 years old) performed two trials of fatiguing isometric hand-grip exercise followed by PEMI, in control conditions and with end-tidal CO2 (P ET ,CO2) clamped at ∼1 mmHg above the resting partial pressure. In the control trial, P ET ,CO2 decreased from rest during hand-grip exercise and PEMI, while MCA Vmean was unchanged from rest. By design, P ET ,CO2 remained unchanged from rest throughout the clamp trial, while MCA Vmean increased during hand-grip (+10.6 ±1.8 cm s(-1)) and PEMI (+9.2 ± 1.6 cm s(-1); P < 0.05 versus rest and control trial). Increases in minute ventilation and mean arterial pressure during hand-grip and PEMI were not different in the control and P ET ,CO2 clamp trials (P > 0.05). These findings indicate that metabolically sensitive skeletal muscle afferents play an important role in the regional increase in cerebral perfusion observed in exercise, but that influence can be masked by a decrease in P ET ,CO2 when they are activated in isolation during PEMI.