Exposure of the arterial endothelium to low and disturbed flow is a risk factor for the erosion and rupture of atherosclerotic plaques and aneurysms. Circulating and locally produced proteins are known to contribute to an altered matrix composition at the site of lesions, and to contribute to inflammatory processes within the lesions by altering the sub-endothelial matrix. We have previously shown that immune-cell regulated alternative splicing of Fibronectin (FN) protects against flow-induced hemorrhage. Here, we perform quantitative proteomic analysis of enriched extracellular matrix preparations from murine carotid arteries exposed to low and disturbed flow in vivo and examine serum derived and endothelial cell contributions to the sub-endothelial matrix in vitro. Our results reveal the extent of the dynamic alterations in extracellular matrix composition in the acute response to low and disturbed flow, and show how changes in the splicing of FN, a common response in vascular inflammation and remodeling, affects matrix composition.