This experiment examined the effect of length of exposure of bovine M. longissmus dorsi to high temperatures (37°C) on proteolytic activity during post-mortem aging and subsequent meat tenderness. To avoid confounding between pH decline and incubation treatments, the experiment was conducted on post-rigor meat (pH < 5.6), which had entered rigor at 15°C. This meat was first incubated at 15°C until rigor (pH < 5.6), whereupon samples were then reheated and maintained at 37°C for 0, 1 or 3 h. Following incubation, samples were aged at 1°C for 1–21 days. Control groups were incubated at either 15 or 37°C until pH < 5.6, followed by aging at 1°C. High rigor temperatures accelerated post-mortem proteolysis early in the post-mortem period, as indicated by a rapid decline in shear force (P < 0.01), but post-mortem aging ceased at about day 3 post-mortem and the high rigor temperature treatment was ultimately 30% tougher at day 21 post-mortem (P < 0.01). The process of reheating samples from 15 to 37°C had minimal effect on tenderness levels, but was associated with a slight increase in proteolysis, identified by myofibril length, and was associated with an increase in cook loss percentage (P < 0.05). Shear force in the length of exposure experiment was affected by an incubation time × post-mortem aging interaction (P < 0.01). This indicated an initial acceleration of post-mortem aging with increased length of exposure, but also a reduction in the ultimate tenderness of product after extended post-mortem aging. This was presumably due to the loss of proteolytic enzyme activity caused by the instability of proteolytic enzymes at high ionic strength conditions such as those present in post-mortem muscle.