Free VWF association and dissociation from tethered VWF was measured in real time at the single-molecule level with a pressure-actuated microfluidic flow cell attached to a TIRF microscope. VWF concatemers were initially labeled with biotin and Alexa 488 (green) and immobilized on the traptavidin-coated flow cell wall (tethered VWF). A second fraction of VWF, labeled with Alexa 647 (purple), flowed through the microfluidic cell at different rates. High-flow rates resulted in high tensile forces proximal to the tether points, which are necessary for VWF self-association.