We have recently demonstrated that the use of a tourniquet to locate the vein prior to blood sampling may affect red blood cell (RBC) deformability and aggregation, even if the tourniquet is quickly removed after insertion of the needle into the vein. However, in clinical practice, the tourniquet is usually kept in place until blood sampling is completed. The present study tested the effects of tourniquet application during the blood sampling period on RBC deformability and aggregation. We compared blood samples obtained at 5, 30, 60 and 90 seconds following the tourniquet application; a control blood sample was obtained without applying a tourniquet. RBC deformability was slightly increased at 5 seconds (7.6% mean, 1–5 Pa) and at 60 seconds (5.9% mean, 1–30 Pa) but not at 30 or 90 seconds, with the increase inversely related to shear stress. The RBC aggregation index was slightly but significantly decreased at 5, 30 and 60 seconds, with no changes observed for either amplitude or half-time of aggregation. These very modest yet significant effects of tourniquet application on hemorheological parameters may not be meaningful in terms of pathophysiology, but may become important in data interpretation or for specific subjects.