The osmotic fragility test (OFT), conventionally used for assisting the diagnosis of many erythrocyte disorders, is a manual and time-consuming analysis not daily performed in many medical laboratories. This study was aimed at defining the stability of whole blood samples used for assessing erythrocyte osmotic resistance.Twenty-one consecutive routine whole blood samples collected into 5.4 mg K2EDTA were tested immediately after collection (day 0) and at different time intervals afterward (day 1, 2, 3, 4, 7, 10 and 14) after storage at 4 °C. The OFT was performed with the Osmored Monotest (1.3% glycerol; Eurospital, Trieste, Italy). Results at the different time points were compared with those obtained at day 0 and with the reference change value (i.e., 33%).The median value of both hyperosmolar and hyposmolar resistance increased from baseline, reaching statistical significance at day 7 for hyperosmolar resistance and at day 1 for hyposmolar resistance, respectively. The median relative increase of hemolysis percentage values become greater than the reference change value at day 3 for hyposmolar resistance, while this limit was never overcome for hyperosmolar resistance. A significant inverse association was found between the mean increase in hyperosmolar resistance and the baseline value of hyperosmolar resistance (r = -0.92), mean corpuscular volume (MCV; r = -0.46) or mean corpuscular hemoglobin (MCH; r = -0.44), as well as between the mean increase in hyposmolar resistance and the baseline value of hyposmolar resistance (r = -0.86), or patient age (r = -0.56).The sample stability seems critical for the OFT. Whole blood specimens should not be stored refrigerated at 4 °C for >2 days before testing.