<p>Ultrasound assisted marination is known to accelerate NaCl diffusion, modify protein structures, and improve the texture of meat products under low-temperature conditions. Multi-frequency ultrasound offers high processing efficiency and versatile mode combinations. Therefore, this study investigated the effects of various multi-frequency ultrasound modes on NaCl diffusion and the structure of myofibrillar proteins (MPs) in beef tenderloin during low-temperature marination. A predictive model based on Fick’s second law was developed and complemented by COMSOL simulations to analyze the salt diffusion process. Concurrently, the impact of these ultrasonic modes on MPs structure was examined to elucidate the underlying mechanism for the accelerated marination. Results showed that the mathematical model accurately predicted salt content changes, while the numerical simulation confirmed uniform and rapid salt diffusion. While the ultrasonic treatments did not significantly alter the primary structure of MPs, they markedly increased the myofibril fragmentation index. Furthermore, marination under multi-frequency ultrasound induced a conformational shift in the secondary structure of the proteins, characterized by a decrease in α-helices and an increase in β-sheets. This structural alteration along with significant fragmentation of the MPs microstructure and evidence of protein degradation, facilitated accelerated salt penetration. This research provides a fundamental basis for the mechanism of ultrasound-assisted marination and supports the development of advanced marination technologies for enhancing meat processing efficiency.</p> Graphical Abstract <p></p>