Red blood cell (RBC) deformability may increase, or decrease, following application of shear stress ("shear conditioning"), depending upon the specific magnitude and duration of exposure. However, the time course of altered RBC deformability following shear remains unresolved. We utilised shear conditioning known to increase (10 Pa) or decrease (64 Pa) RBC deformability and subsequently rested the cells; serial measurements of deformability during the rest period facilitated defining the time course of recoverability. A second experiment repeated the shear conditioning and recovery period to explore whether multiple duty-cycles augmented the response following the initial exposure. Shear conditioning was performed for 300 s at the desired shear stress. Ektacytometry was used to quantify human RBC deformability immediately and during rest (3, 5, 60, 120, 240, 300 s) using discrete samples. RBC were shear conditioned twice in a separate experiment, with 300 s rest separating the conditioning. Shear conditioning at 10 Pa induced increased cell deformability by 19.5 ± 0.3%, which reduced to 7.2 ± 0.4% after 300 s of rest. Shear conditioning at 64 Pa decreased cell deformability by 30.5 ± 13.9%, and after 300 s rest, remained decreased (19.3 ± 9.4%) compared with baseline. The second duty-cycle augmented initial responses induced by shear conditioning. Specific shear conditioning results in either temporarily increased cell deformability, or a less reversible decrease of RBC deformability.