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ABSTRACT: The effect of ultrasound on Biceps femoris muscle tenderness was investigated using a 22 Central Composite Rotatable Design (CCRD) with triplicates at the central point. We evaluated the following independent variables: ultrasound intensity ranging from 11.30 to 33.90 W cm-2 and exposure time between 35 and 205 s. The ultrasound bath’s frequency (80 kHz) and temperature (10 ºC) were the fixed ones. To validate the model, the muscle was treated at the CCRD’s optimized condition (80 kHz, 22.60 W cm-2, 120 s, 10 ºC) evaluated, and compared with the muscle control sample (non-treated). A 22% shear force reduction was observed compared to the control sample (no ultrasound treatment) after 144 h, and stored at 5 ºC. Moreover, a sarcoplasmic calcium concentration increase was noted for ultrasound-treated muscle, probably activating the calpain enzyme system. In contrast, no significant influence (P > 0.05) was observed for pH, color index, lipid oxidation, water holding capacity, and drip loss by ultrasound treatment at the optimized conditions. Therefore, ultrasound application is promising and suitable for improving muscle tenderness without losing meat quality. This study highlighted the ultrasound effect on the tenderness of a less studied muscle (Biceps femoris) by combining short ultrasound exposure (120 s) and an 80 kHz frequency.