Tenderness is a major factor in consumer perception and acceptability of beef meat. Here we used a laboratory tumbling simulator to investigate the effectiveness of the tumbling process in reducing the toughness of raw beef cuts. Twelve Semitendinosus beef muscles from cows were tumbled according to four programs: T1 (2500 consecutive compression cycles (CC), for about 3 h), T2 (6000 CC, about 7.5 h), T3 (9500 CC, about 12 h), and T4 (13,000 CC, about 16 h). The effect of tumbling on the toughness of raw meat was assessed using compression tests (stresses measured at 20% and 80% of deformation ratios) and microscopic observations made at the periphery and centre of meat samples, and compared against non-tumbled controls. Longer tumbling times significantly reduced the stresses measured at 20% and 80% compression rates, which reflected the toughness of muscle fibres and connective tissue, respectively. At the microscopic level, longer tumbling times led to reduced extracellular spaces, increased degradation of muscle structure, and the emergence of amorphous zones. A 12-h tumbling protocol ultimately makes the best compromise between the process time demand and toughness reduction in beef Semitendinosus meat pieces.