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Accelerometers are increasingly used to observe human behavior such as physical activity under free-living conditions. An important prerequisite to obtain reliable results is the correct calibration of the sensors. However, accurate calibration is often neglected, leading to potentially biased results. Here, we demonstrate and quantify the effect of accelerometer miscalibration on the estimation of objectively measured physical activity under free-living conditions. The total volume of moderate to vigorous physical activity (MVPA) was significantly reduced after post hoc auto-calibration for uniaxial and triaxial count data, as well as for Euclidean Norm Minus One and mean amplitude deviation raw data. Weekly estimates of MVPA were reduced on average by 5.5, 9.2, 45.8, and 4.8 min, respectively, when compared to the original uncalibrated estimates. Our results indicate a general trend of overestimating physical activity when using factory-calibrated sensors. In particular, the accuracy of estimates derived from the Euclidean Norm Minus One feature suffered from uncalibrated sensors. For all modalities, the more uncalibrated the sensor was, the more MVPA was overestimated. This might especially affect studies with lower sample sizes. [ABSTRACT FROM AUTHOR]