Background: Cerebral blood flow and reactivity declines with older age, which increases the risk of cerebrovascular disease and cognitive decline. Cerebral disease is also associated with reduced vascular function and elevated blood pressure. Isometric handgrip (HGEx) training is emerging as an alternative approach for improving vascular function. However, the effect of acute HGEx on cerebral blood flow and function, including endothelial function, is not well understood. Cerebral flow mediated dilation (cFMD) is suggested as a novel and recent technique that provides an index of extracranial cerebrovascular endothelial function. This study aimed to assess the acute effect of isometric handgrip exercise on cFMD and intracranial cerebrovascular reactivity of the middle cerebral artery velocity (MCAv CVR) in middle-aged healthy adults. Methods: Twelve middle-aged, healthy adults (8 females, age 54 ± 7.8 years; BMI 26±3.5 kg/m2) performed isometric unilateral HGEx at 30% of maximal voluntary effort for three minutes. Intracranial (MCAv) and extracranial blood flow of the internal carotid artery (ICA) were measured continuously by Transcranial Doppler and Duplex ultrasound, respectively. At baseline and immediately following HGEx, cFMD and MCAv CVR was assessed using a hypercapnic breathing challenge (5% CO2). Beat-by-beat blood pressure and cardiac output, heart rate and end-tidal of carbon dioxide (PETCO2) were measured continuously throughout HGEx, cFMD and CVR assessments. Cerebral blood flow,blood pressure and PETCO2, was analysed using a custom built edge detection and graphing software. Statistical analyses were performed using RStudio. All data are expressed as mean ±SD unless otherwise stated. Hemodynamic responses during HGEx are displayed as change from baseline (%). Results: Resting mean arterial pressure was 89±10 mmHg, and resting heart rate was 56±7 bpm. HGEx significantly increased extracranial and intracranial cerebral blood flow (ICA diameter, 4.4±4.7%, p=0.025; shear rate, 28.2±18.3%, p≤ 0.001; MCAv, 24.9±21%, p≤ 0.005) and blood pressure (MAP 19.9±10.2%), p≤ 0.001). cFMD was higher following HGEx but this was not statistically significant (cFMD pre/ post: 4.7± 3.3 % vs. 6.7± 2.9 %, p= 0.11). MCAv reactivity was significantly higher following HGEx (CVR pre/ post: 16.3±14.4% vs 29.5±11.4%, p<0.001). Conclusion: These preliminary data reveal that isometric HGEx may be an effective exercise mode to promote acute increases in CVR. Our findings also support future larger investigations into the benefits of HGEx for improving cFMD. The effect of a HGEx intervention on cerebrovascular function is warranted.