Individuals who experience prolonged sitting daily are reported to be at risk of developing cerebrovascular disease, which is associated, in part, with attenuation in cerebral blood flow regulation. However, the effect of prolonged sitting on dynamic cerebral autoregulation (dCA), a crucial mechanism of cerebral blood flow regulation, remains unclear. Additionally, cerebrovascular disease occurs heterogeneously within cerebral arteries. The purpose of the present study was to examine the hypothesis that prolonged sitting attenuates dCA in the cerebral circulation heterogeneously. Twelve young, healthy participants were instructed to maintain a seated position for 4h without moving their lower limbs. Mean arterial pressure and mean blood velocities of the middle cerebral artery (MCA Vm) and the posterior cerebral artery (PCA Vm) were measured continuously throughout the experiment. The dCA was assessed using transfer function analysis (TFA) with mean arterial pressure and either MCA Vm or PCA Vm. In the MCA, very low-frequency TFA-normalized gain decreased significantly during 4h of prolonged sitting (P=0.029), indicating an improvement rather than attenuation in dCA, despite a significant reduction in MCA Vm after 4h of continuous sitting (P=0.039). In the PCA, PCA Vm remained stable throughout the 4h sitting period (P=0.923), and all TFA parameters remained unchanged throughout the 4h of sitting. Contrary to our hypothesis, these results suggest that the dCA in both the MCA and the PCA was well stabilized in healthy young individuals during acute prolonged sitting.