Introduction: Evidence indicates that the cerebral vessels is subjected to sympathetic nerve activity (SNA), yet whether or not human cerebral blood flow (CBF) is affected by SNA remains controversial. It has been observed that high-intensity exercise induced fatigue of the respiratory muscles can initiate a metaboreflex, causing sympathoexcitation and vasoconstriction in the limbs, thereby limiting limb blood flow and O2 delivery to both the resting and exercising locomotor muscles. However, it is not known whether fatigue of the respiratory muscles can induce vasoconstriction of the cerebral vasculature and thus attenuate CBF and cerebral oxygenation. The aim of the present study was to control the influence of the arterial partial pressure of CO2 on CBF, by clamping end-tidal PCO2 (PETco2) to isocapnic levels during high-intensity exercise, in order to examine the possible influence of a respiratory metaboreflex on CBF and cerebral oxygenation. Methods: Twelve endurance-trained males participated in this cross-over study. Subjects visited the laboratory 3 times in total; 1 for familiarization including determination of maximal oxygen consumption (VO2max), and 2 for either a control (CON) or isocapnic trial (ISO), consisting of constant load high-intensity (≥85% VO2max) cycling to exhaustion. Mean blood flow velocity in the middle cerebral artery (MCA vmean) and frontal lobe cerebral oxygenation (ScO2) were measured during both trials with transcranial Doppler ultrasound and NIRS, respectively. To confirm the presence of respiratory muscle fatigue, maximal inspiratory pressure (MIP) was measured pre- and post-exercise. Results: Mean exercise duration and intensity during the trials were 12 min 24.9 s ± 1min 18 s and 91 ± 5 %VO2max, respectively. During ISO Petco2 was successfully clamped at 40 ± 1 mmHg. The presence of respiratory muscle fatigue was confirmed by a 12% (P < 0.001B) decrease in ISO and a 7% (P = 0.025B) decrease in CON of MIP. MCA vmean increased 12% in ISO from clamping to end-exercise (P = 0.017B), while it remained unchanged during CON. Similar, at end-exercise MCA vmean was higher during ISO than CON (P < 0.002B). ScO2 decreased in both ISO (P = 0.001B) and CON (P < 0.001B) at end-exercise, with no difference between trials. Discussion: Although respiratory muscle fatigue was induced, we could not confirm the presence of a respiratory muscle fatigue induced decrease in MCA vmean during ISO. In contrast, a decrease in ScO2 was observed despite the increased MCA vmean. Thus, it cannot be excluded that a SNA induced decrease in CBF did occur in other arteries, but that it remained undetected, since only flow velocity in MCA was measured. In conclusion, the presence of a respiratory muscle fatigue induced attenuation of CBF during high-intensity exercise could not be confirmed. Because the regulation of CBF is multifactorial, and this study only measured MCA vmean, and controlled Paco2, by means of clamping PETco2, it cannot be excluded that a respiratory metaboreflex contributes to an overall SNA-induced regulation of CBF.