Viscosity of the suspending medium can be a determinant contributor to the effective measurements of red blood cell deformability using ektacytometry. Deformability of erythrocyte is known to show inter-species and gender differences, therefore the evaluation of the effects and the standardized usage of the suspending media are important in animal experiments. For evaluating the differences using various viscosity media, we aimed to test male and female Sprague-Dawley rats and beagle dogs. Blood samples were collected and red blood cell deformability was tested by a RheoScan-D200 slit-flow ektacytometer using parallel measurements in PVP solutions at viscosity of 15, 20 and 30 mPa.s. The lowest elongation index values were measured in 15 mPa.s solution in both species and genders. The inter-species differences were obvious: rats had higher elongation index values. Gender differences were found only in 20 and 30 mPa.s samples: in rats males, while in dogs females had better red blood cell deformability. The standard usage of PVP solutions is essential for comparative studies, because the results obtained from different viscosity PVP solutions are not comparable to each other. In Sprague-Dawley rats and beagle dogs the relatively less viscous media (<20 mPa.s) may result in less informative data.