Tenderness, water loss, colour and lipid stability are important quality attributes of fresh meat. This study investigated the effect of repeated (1×, 2× or 3×) pulsed electric field treatment (10 kV, 90 Hz, 20 μs) on the tenderness, purge loss, cooking loss, colour, lipid oxidation, protein profile and post-treatment proteolysis of cold-boned beef longissimus lumborum and Musculus semimembranosus muscles. The shear force of beef longissimus lumborum was found to decrease by 2.5 N with every extra application of pulsed electric field treatment, while the shear force of M. semimembranosus muscle was not significantly affected by pulsed electric field treatment. There was an increase in proteolysis of beef longissimus lumborum treated with 1× pulsed electric field as evident by increased troponin T and desmin degradation; however, less degradation was found with increasing number of pulsed electric field treatments suggesting another mechanism such as physical disruption is responsible for the tenderisation of beef by pulsed electric field.