ABSTRACT Overall, meat texture is composed of two types of toughness: primary toughness, because of mechanical resistance of the myofibrillar structure, and secondary toughness, by reason of connective tissue content. Primary toughness can be reduced during aging by intrinsic and extrinsic protease activity. Secondary toughness is determined by a given amount of epimysial and perimysial connective tissue, which cannot be reduced as no collagenases are naturally present in the muscle or produced by native meat microbial populations. The objective of this work was to study primary toughness reduction in meat from four animal species, i.e., beef, horse, rabbit and hen, employing M. Biceps femoris, by calpain activation with calcium chloride. Secondary toughness, expressed as hydroxyproline content, and contribution of both parameters to overall hardness were also studied. Marination with 150 mM CaCl 2 increased enzymatic activity in pre‐rigor meat (horse and beef), but higher concentrations (250 mM) reduced enzymatic activity increments. Although beef marination considerably increased enzymatic activity, it did not reduce hardness, probably because of high collagen content. The high hydroxyproline concentration in beef and horse caused an overall hardness. Marinated horse meat had lower collagen content and a higher enzyme activity, resulting in less hard samples. As expected, pre‐rigor meats, hen and rabbit, had low hardness values for both control and treated samples. Because CaCl 2 in high concentrations inhibits calpain activity, it was assumed that rabbit meat calpains were inhibited at concentrations as low as 150 mM. Therefore, calpains were active in control pre‐rigor rabbit meat but were inactivated by CaCl 2 addition.