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Objective: Physical loading changes bone microstructure and may influ-ence quantitative ultrasound (QUS) parameters. This study aims at evaluating the effect of physical loading on bone QUS measurement. Methods:Ten fresh bovine bone specimens were studied, which were scanned by a micro-CT and the density and structure parameters were calculated. The QUS measurement was performed when specimens sub-jected to loading, which changed from 0 to 1,000 N with the step of 100 N. Statistical analysis was performed to evaluate the difference between nBUA measured with and without loading, and the relationship between nBUA and the parameters measured by micro-CT. Results:While the loading exerted on bone lugher than 200 N, the measured nBUA significantly higher than nBUA measured without loading. With the increasing of loading, which exerted on bone, the values of nBUA also increased. A new parameter, the slope of the linearity fitted curve of nBUA values measured under different loading conditions, was introduced to evaluate BMD. The correlation coefficient between the slope and BMD is -0.869 (P=0.001). Conclusion: Physical loading substantially influences bone QUS measurement. QUS measurement performed under loading condition may be a new ultrasound method for osteoporosis diagnosis.