Detection of semen storage-induced alterations in preserved boar spermatozoa is crucial to assess fertilization capacity. Multicolor flow cytometry now offers the possibility for simultaneous measurement of multiple capacitation markers in liquid stored boar spermatozoa based on single cell evaluation. The aim of the present study was to establish a novel multicolor capacitation assay and to determine its sensitivity to detect storage-induced functional changes in boar sperm physiology. Sperm analysis was performed in semen doses (n = 66 boars) diluted in Beltsville Thawing Solution after 72 h and 96 h of storage at 17°C. Motility (AndroVision®, Minitube) and integrity of plasma membranes and acrosomes (flow cytometry: Propidium iodide/ FITC-PNA) differed only slightly between the storage time points (motility: 76.2 ± 9.9 vs. 71.6 ± 18.2; membrane integrity: 81.8 ± 7.9 vs. 79.3 ± 11.2; p<0.05). For multicolor visualization of capacitation-ability, sperm were loaded with flourochromes Yo-Pro-1, Merocyanine (M) 540 and Calbryte (Cbry) 630. The flow cytometer CytoFLEX (Beckman Coulter), equipped with three lasers (488 nm, 638 nm, 405 nm) and 13 filters was used. Semen doses were incubated in Tyrode’s capacitating medium under CO2 at +38°C or in a non-capacitating control medium (Tyrode w/o bicarbonate) and analyzed after 3 and 60 min incubation time. Storage did not influence the percentage of viable (Yo-Pro neg.) sperm with high membrane fluidity (M540 pos.) and high intracellular calcium (Cbry pos.) under non-capacitating conditions. After 60 min in capacitation medium, semen stored for 96 h showed a lower percentage of M540 pos. and of Cbry pos. viable spermatozoa compared to 72 h (p < 0.05). This corresponded to a lower specific response to the capacitation stimulator bicarbonate based on the decrease of viable, M 540 neg. and Ca2+ neg. spermatozoa at 96 h (47.8 ± 22.6 %) compared to 72 h (54.8 ± 18.6 %; p < 0.01) of storage. In conclusion, multicolor flow cytometry assessment of capacitation-induced changes is a sensitive tool to detect subtile storage effects in viable sperm. Although the biological relevance in AI setting remains to be shown, such assays are helpful to optimize semen preservation techniques.