The stability of μ-calpain, m-calpain and calpastatin activity during frozen storage of pork was studied in two experiments. In experiment 1, pork longissimus muscle was stored at either -20 or -80°C, and the samples were assayed at 2-3 weeks interval for calpain activity and calpastatin activity using a m-calpain stock solution stored at 4°C. No effects on calpain activity at either temperature were observed for up to 123 days of storage. Calpastatin activity was stable the first few weeks of storage, where after it decreased up to 143 days of storage independently of meat storage temperature. At day 143, calpastatin activity was also assayed using a newly purified stock solution of m-calpain giving a calpastatin activity equal to the activity measured day 0 using the original m-calpain stock solution. The m-calpain stock solution was unstable during storage at 4°C and the activity decreased in a linear manner and was highly related to the observed decrease in calpastatin activity during storage. In experiment 2, meat was stored as in experiment 1 and was assayed at 2-3week intervals for calpastatin activity using a m-calpain stock solution stored at either 4 or -80°C. As in experiment 1, the measured activity of calpastatin decreased during storage using m-calpain stock solution stored at 4°C and this decrease was highly correlated to the decrease in the activity of the m-calpain stock solution. The activity of the m-calpain stock solution stored at -80°C was constant during storage period of 153 days and likewise was the calpastatin activity measured using this stock solution. The relation between measured calpastatin activity and storage time of m-calpain stock solution was tested by adding, to a calpastatin assay, up to 10μL of a partly inactivated m-calpain solution. A negative relationship was observed between added inactivated m-calpain and measured calpastatin activity which suggests that the inactive m-calpain molecules mask some of the binding sites on calpastatin and thereby prevent some of the active m-calpain molecules from binding to calpastatin. This would underestimate the measured calpastatin activity. In conclusion, the calpains as well as calpastatin are stable during frozen storage of meat, and the observed decreased in calpastatin activity is due to instability of the m-calpain stock solution used in the calpastatin assay.