Background: Harmful health effects associated with sedentary behavior may be attenuated by breaking up long periods of sitting by standing or walking. However, studies assess interruptions in sitting time differently, making comparisons between studies difficult. It has not previously been described how the definition of minimum break duration affects sitting outcomes. Therefore, the aim was to address how definitions of break length affect total sitting time, number of sit-to-stand transitions, prolonged sitting periods and time accumulated in prolonged sitting periods among office workers. Methods: Data were collected from 317 office workers. Thigh position was assessed with an ActiGraph GT3X+ fixed on the right thigh. Data were exported with varying bout length of breaks. Afterward, sitting outcomes were calculated for the respective break lengths. Results: Absolute numbers of sit-to-stand transitions decreased, and number of prolonged sitting periods and total time accumulated in prolonged sitting periods increased, with increasing minimum break length. Total sitting time was not influenced by varying break length. Conclusions: The definition of minimum break length influenced the sitting outcomes with the exception of total sitting time. A standard definition of break length is needed for comparison and interpretation of studies in the evolving research field of sedentary behavior.