To assess the influence of rheological properties of an artificial vitreous (AV) on the performance of double-blade (DB) and single-blade (SB) guillotine vitreous cutters, with 23-, 25-, and 27-gauge (G) probes. We evaluate the aspiration flow rate, using an optical method, based on image processing. Experiments are conducted using ten viscoelastic vitreous phantoms, with different properties that are measured with rheological tests. Aspiration rate strongly varies with fluid properties. Regardless of cutter geometry and operational conditions, the flow rate significantly decreases as vitreous viscosity and elasticity increase. All tested vitreous probes are very sensitive to changes in fluid rheology. SB cutters produce smaller flow rates compared with DB ones of the same caliber; however, they are less sensitive to fluid properties at low aspiration pressures. The use of vitreous substitutes for test performance guarantees comparability between flow rate results achieved with different vitrectomy systems operating in different media. This outcome is further confirmed by the low values of estimated flow rate relative errors.