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Low-intensity pulsed ultrasound (LIPUS) has been proposed for novel therapies still under study, where similar parameters and protocols have been used for producing opposite effects that range from increasing cell viability to provoking cell death. Those divergent outcomes make the gener-alization of expected effects difficult for cell models not yet studied. This paper presents the effect of LIPUS on the viability of the MIO-M1 cell line for two well-established setups and different pro-tocols; the acoustic intensities, duty factors, and treatment duration were varied. Measurements and models for acoustic and thermal analysis are included for proposing a solution to improve the reproducibility of this kind of experiments. Results indicate that MIO-M1 viability is less affected for the cells treated through a dish that is partially immersed in water; in these conditions, the cells neither show detrimental nor proliferative effects at intensities lower than 0.4 W/cm2 at 20% duty factor. However, cell viability was reduced when LIPUS was followed by cell subculturing. Treating the cells through a gel, with the culture dish placed on the transducer, increases cell mor-tality by the production of standing waves and mixed vibration-acoustical effects. Using the wa-ter-based setup with a 1° dish inclination reduces the effects of standing waves.