There is conflicting evidence regarding the health implications of high occupational physical activity (PA). Shoe-based accelerometers could provide a feasible solution for PA measurement in workplace settings. This study aimed to develop calibration models for estimation of energy expenditure (EE) from shoe-based accelerometers, validate the performance in a workplace setting and compare it to the most commonly used accelerometer positions. Models for EE estimation were calibrated in a laboratory setting for the shoe, hip, thigh and wrist worn accelerometers. These models were validated in a free-living workplace setting. Furthermore, additional models were developed from free-living data. All sensor positions performed well in the laboratory setting. When the calibration models derived from laboratory data were validated in free living, the shoe, hip and thigh sensors displayed higher correlation, but lower agreement, with measured EE compared to the wrist sensor. Using free-living data for calibration improved the agreement of the shoe, hip and thigh sensors. This study suggests that the performance of a shoe-based accelerometer is similar to the most commonly used sensor positions with regard to PA measurement. Furthermore, it highlights limitations in using the relationship between accelerometer output and EE from a laboratory setting to estimate EE in a free-living setting.