The increase in cerebral perfusion observed during isometric handgrip (IHG) is abolished by removal of skeletal muscle afferent feedback with regional anesthesia. However, middle cerebral artery velocity (MCA Vmean) is unchanged from baseline during post-exercise muscle ischemia (PEMI) where the stimulation of metabolically sensitive skeletal muscle afferents (muscle metaboreflex) is isolated. We tested the hypothesis that a hyperventilation-mediated decrease in the partial pressure of arterial carbon dioxide (CO2), and hence augmented cerebral vasoconstriction, masks the effect of muscle metaboreflex stimulation on MCA Vmean during PEMI. 10 healthy men (20±1 yr; mean±SEM) performed bouts of fatiguing IHG at 40% maximal voluntary contraction followed by PEMI, under control conditions and with end-tidal carbon dioxide (PETCO2) clamped at ≈1 mmHg above baseline on a breath-by-breath basis. IHG bouts were separated by a 30 min rest period and conducted according to a single blind randomized crossover design. MCA Vmean (transcranial Doppler), blood pressure (BP; Portapres), heart rate (HR; electrocardiogram) and minute ventilation (VE; turbine) were continuously recorded throughout. Main effects of time (baseline, 25, 50, 75 and 100% of IHG duration, PEMI), condition (control, PETCO2 clamp) and their interaction were investigated with two-way repeated measures ANOVA and Holm-Sidak post hoc tests. The effect of condition on IHG duration was examined using a paired t-test. IHG duration was not different in the control and PETCO2 clamp conditions (118±7 vs. 128±11 s, respectively; P>0.05). In the control condition, PETCO2 decreased progressively from baseline (39.3±0.7 mmHg) during IHG (-4.8±1.4 mmHg from baseline at 100% IHG; P<0.05) and PEMI (-6.6±1.4 mmHg from baseline; P<0.05), while MCA Vmean was not significantly different from a baseline of 62.5±2.4 cm.s-1 at 100% IHG and PEMI (+3.1±4.7 and -3.6±2.5 cm.s-1 from baseline, respectively). By design, PETCO2 remained unchanged from baseline in the clamp condition, and while MCA Vmean was not significantly altered at baseline (65.6±2.7 cm.s-1; P>0.05 vs. control) a notable increase of +10.6±1.8 and +9.2±1.6 cm.s-1 was observed at 100% IHG and PEMI (P<0.05 vs. baseline and control). VE, mean BP and HR were not different at baseline, IHG and PEMI during the control and PETCO2 clamp conditions (P>0.05). VE and mean BP were elevated from baseline during IHG and PEMI (P<0.05), while HR was only increased during IHG (P<0.05). Thus, clamping PETCO2 at baseline levels during PEMI reveals a muscle metaboreflex mediated increase in MCA Vmean, suggesting that a hyperventilation-mediated decrease in the partial pressure of arterial CO2 can mask the effect of muscle metaboreflex stimulation on cerebral perfusion.