Objective: This pilot study explores the influence of acute alcohol exposure on cell mechanical properties of steady-state and activated leukocytes conducted with real-time deformability cytometry. Methods: Nineteen healthy male volunteers were enrolled to investigate the effect of binge drinking on biophysical properties and cell counts of peripheral blood leukocytes. Each participant consumed an individualized amount of alcohol to achieve a blood alcohol concentration of 1.2 ‰ as a mean peak. In addition, we also incubated whole blood samples from healthy donors with various ethanol concentrations and performed stimulation experiments using lipopolysaccharide and CytoStim™ in the presence of ethanol. Results: Our findings indicate that the biophysical properties of steady-state leukocytes are not significantly affected by a single episode of binge drinking within the first two hours. However, we observed significant alterations in relative cell counts and a shift toward a memory T cell phenotype. Moreover, exposure to ethanol during stimulation appears to inhibit the cytoskeleton reorganization of monocytes, as evidenced by a hindered increase in cell deformability. Conclusion: Our observations indicate the promising potential of cell mechanical analysis in understanding the influence of ethanol on immune cell functions. Nevertheless, additional investigations in this field are warranted to validate biophysical properties as biomarkers or prognostic indicators for alcohol-related changes in the immune system.