The objective of this study was to evaluate the importance of proteolysis and sarcomere length in determining the tenderness of bovine M. longissimus dorsi (LD) muscle over a 21-day period. This was done by altering the pre-rigor glycolytic behaviour of hot-boned LD muscles using different early post-mortem temperature regimes. Hot-boned LD muscles (n=8) were cut in two, randomised, placed in polythene bags and submerged in a water bath set at 5°C (rapidly chilled) and 15°C (slowly chilled) for 8h post-mortem. All muscles were then stored at 2°C for up to 21 days post-mortem. The temperature regimes altered the glycolytic behaviour of the muscles in the pre-rigor period. The slowly chilled muscles exhibited a faster (P<0.01) pH fall than rapidly chilled muscles. Cold shortening was induced in rapidly chilled muscles as they had shorter (P<0.01) sarcomere lengths than slowly chilled muscles up to day 21 post-mortem. Warner Bratzler shear force values (WBSF) deemed cold-shortened muscles as tougher than noncold shortened up to day 14 post-mortem. Both cold-shortened and noncold-shortened muscles tenderised over time to an extent where there was no significant difference in WBSF values by day 21 post-mortem. SDS–PAGE protein profiles indicated that the rate of proteolysis was faster in slowly chilled muscles when compared to rapidly chilled muscles. However by day 21 post-mortem it appeared that rapidly and slowly chilled muscles underwent proteolysis to the same extent.