Ischemic stroke has been shown to induce breakdown of the blood-brain barrier, although these changes are not fully characterized. Oxygen-glucose deprivation (OGD) has been used to investigate the effects of ischemia in cultured brain capillary endothelial cells, however this involves a change of medium which in itself may affect the cells. The aim of the present study was to investigate the effect of OGD and simple medium exchange followed by 48 h of reperfusion on barrier properties of primary bovine endothelial cells co-cultured with rat astrocytes. Barrier properties were evaluated by transendothelial electrical resistance measurements, passive permeability of flux markers, RT-qPCR and immunocytochemistry. Both OGD and simple medium exchange caused an increase in endothelial monolayer permeability. This correlated with reduced transcript levels of a number of tight junction and tight junction-associated proteins (claudin-1, claudin-5, occludin, ZO-1, tricellulin, marveld3 and PECAM-1), as well as with altered transcript level of several transporters and receptors (GLUT-1, HB-EGF, InsR, TfR, two members of the low density lipoprotein receptor family, LDLR and LRP-1, and the efflux transporter BCRP). In contrast, effects induced specifically by OGD were transient de-localization of claudin-5 from the junction zone, increased InsR localization at the plasma membrane and transient downregulation of MRP-1 and P-gp transcript levels. In conclusion, OGD caused changes in claudin-5 and InsR localization, as well as in MRP-1 and P-gp transcript levels. Our results however also indicated that medium exchange alone caused changes in functional barrier properties and expression levels of wide range of proteins.