Abstract The effect of extraction buffer on extractable calpain and calpastatin activity in postmortem muscles was examined. Muscles were removed from ovine carcasses 24 h after slaughter and extracted with three volumes of two extraction buffers containing 20 (pH 7.5) and 100 (pH 8.3) mM Tris. There was a significant difference in pH of the muscle homogenates, having a pH of 5.84 and 7.58 for 20 and 100 mM Tris base, respectively. Calpastatin, μ-calpain and m-calpain all had significantly reduced activity in extracts made with 20 mM Tris (pH 7.5) buffer compared to 100 mM Tris (pH 8.3) buffer, showing a loss of, respectively, 30, 57 and 37%. These results indicate the impact of choice of buffer on the extractable calpains and calpastatin activity from postmortem muscle. To avoid loss of calpains due to isoelectric precipitation, the pH of the muscle homogenate (after homogenization and prior to the first centrifugation) must be above 6.2.