Boar spermatozoa are sensitive to storage temperatures below 15 °C. Chilling injury causes loss of motility and membrane integrity in a minority of cells, whereas the main population displays sublethal changes compromising fertility. In this study, changes of the response to capacitation conditions in hypothermically stored boar spermatozoa have been examined using a kinetic approach with well-defined test and control media. Ejaculates of seven boars were diluted in Beltsville Thawing Solution kept for 3 h at 22 °C or cooled to 17, 10 and 5 °C and stored for 24 and 96 h. At each time point, the standard sperm parameters motility and membrane integrity were evaluated. Subsequently, washed subsamples were incubated in capacitating and control medium before flow cytometric analysis of intracellular calcium content using the Fluo-3 probe and changes in phospholipid disorder using merocyanine. Kinetic changes of response parameters were monitored in viable (plasma membrane intact) cells. Chilling led to a loss of standard sperm quality traits in a minor subpopulation of cells, whereas storage length had no effect on these parameters. However, responses to incubation as determined by the loss of live cells with low intracellular calcium content showed marked changes in relation to storage conditions. The specific responsiveness to capacitation conditions decreased in close relation to storage temperature and length. In contrast, the merocyanine probe revealed to be limited to detect effects of hypothermic storage. Using Fourier transform infrared spectroscopy, no influence of chilling on membrane phase behaviour was found that might implicate decreased sperm function. In conclusion, assessment of response to capacitating media by monitoring intracellular calcium levels provides a sensitive measure for chilling injury in extended boar semen, and therefore, deserves implementation in hypothermic storage tests.