Fluid shear stress (FSS) elicits a haemodynamic force on endothelial cells (EC) which can modulate their redox phenotype. Arterial branch points or curvatures where FSS is low and oscillatory (OS) are associated with an “atheroprone” EC phenotype whereas high unidirectional FSS (US) is “atheroprotective” 1. Nuclear factor E2-related factor 2 (Nrf2), a FSS-responsive transcription factor that regulates antioxidant gene expression, is enhanced in response to US2. The endothelial glycocalyx (GCX) is a carbohydrate rich meshwork lining the EC luminal surface. GCX thickness is reduced in models of atherosclerosis and EC exposed to OS3 and is enhanced by US4. Disruption of individual GCX components leads to impaired FSS-mediated nitric oxide production5 highlighting its role in mechanotransduction of FSS to maintain physiological EC function. This study investigates the effects of FSS on antioxidant gene expression and the GCX and also the role of the GCX as a mediator of Nrf2 signalling by US. Human umbilical vein EC (HUVEC) were subjected for 48h to either US (15 dyn/cm2) or OS (±5 dyn/cm2, 1Hz) using microfluidic slides (Ibidi, GmbH) or grown in static conditions. Cells were then fixed (4% formaldehyde) and heparan sulphate (HS), sialic acid (SA) and syndecan-1 (synd-1) GCX components were labelled by immunofluorescence and visualised by confocal microscopy. US and OS (48h) differentially regulated GCX expression compared to static cultures (n=4). Following the same FSS protocols cells were lysed and the Nrf2-regulated antioxidant defence proteins heme-oxygenase 1 (HO-1) and NAD(P)H dehydrogenase quinone 1 (NQO-1) were assessed by western blotting. Total cellular reduced glutathione (GSH), the major intracellular antioxidant, was measured with a fluorometric assay. HO-1 and NQO-1 protein expression were significantly up-regulated at 48h by US compared to static (HO-1 band density units: 0.84±0.07 vs 0.048±0.035, n=5) and to a greater extent (p<0.01) than by OS (0.4±0.08). Total GSH levels were significantly (p<0.05) upregulated by both US (nmol/mg protein: 53.76±3.51) and OS (43.23±3.28) compared to static (32.89±1.6) (n=6). To remove HS or SA components of the GCX, some cells were treated with either heparinase III (HepIII, 50mU/ml, 2h) or Neuraminidase (Neur, 2U/ml, 30min) prior to exposure for 8h to US. HO-1 induction by US was significantly attenuated by Neur (static: 0.07±0.03, US: 1.03±0.06 vs US+Neur: 0.56±0.1, n=5) whereas HepIII had no effect. Values denote mean ± S.E.M., compared by ANOVA. Taken together, these findings demonstrate that the pattern of FSS differentially induces Nrf2 signalling and that the GCX is involved antioxidant gene induction by US, thus strategies to enhance its expression may enhance Nrf2 signalling and promote an antioxidant EC phenotype in regions of disturbed FSS.