OBJECTIVE:The aim of this study was to explore the longitudinal relationship between sitting time on a working day and vitality, work performance, presenteeism, and sickness absence. METHODS:At the start and end of a five-month intervention program at the workplace, as well as 10 months after the intervention, sitting time and work-related outcomes were measured using a standardized self-administered questionnaire and company records. Generalized linear mixed models were used to estimate the longitudinal relationship between sitting time and work-related outcomes, and possible interaction effects over time. RESULTS:A significant and sustainable decrease in sitting time on a working day was observed. Sitting less was significantly related to higher vitality scores, but this effect was marginal (b = −0.0006, P = 0.000). CONCLUSIONS:Our finding of significant though marginal associations between sitting time and important work-related outcomes justifies further research.