Decreased cerebrovascular blood flow and function are associated with lower cognitive functioning and increased risk of neurodegenerative diseases. Prolonged sitting impairs peripheral blood flow and function, but its effects on the cerebrovasculature are unknown. This study explored the effect of uninterrupted sitting and breaking up sitting time on cerebrovascular blood flow and function of healthy desk workers. Fifteen participants (10 male, 35.8{plus minus}10.2 years, BMI: 25.5{plus minus}3.2 kg∙m-2) completed, on separate days, three 4-hr conditions in a randomised order: a) uninterrupted sitting (SIT), b) sitting with 2-min light intensity walking breaks every 30-min (2WALK) or c) sitting with 8-min light intensity walking breaks every 2-hrs (8WALK). At baseline and 4-hrs, middle cerebral artery blood flow velocity (MCAv), carbon dioxide reactivity (CVR) of the MCA and carotid artery were measured using transcranial Doppler (TCD) and duplex ultrasound respectively. Cerebral autoregulation (CA) was assessed with TCD using a squat-stand protocol and analysed to generate values of gain and phase in the very low, low, and high frequencies. There was a significant decline in SIT MCAv (-3.2{plus minus}1.2 cm.s-1) compared to 2WALK (0.6{plus minus}1.5 cm.s-1, p=0.02), but not between SIT and 8WALK (-1.2{plus minus}1.0 cm.s-1, p=0.14). For CA, the change in 2WALK very low frequency phase (4.47{plus minus}4.07 degrees) was significantly greater than SIT (-3.38{plus minus}2.82 degrees, p=0.02). There was no significant change in MCA or carotid artery CVR (p>0.05). Results indicate that prolonged, uninterrupted sitting in healthy desk workers reduces cerebral blood flow, however this is offset when frequent, short-duration walking breaks are incorporated