A strength of the current study is the use of an objective measure to assess sedentary behavior across both weekdays and weekends and relate to a range of mental health outcomes in an adult population. Our findings highlighted that there were weekday and weekend differences in sitting time and that those participants who spent less than 8 hours per day sitting, were less depressed, anxious and had higher levels of vitality than those who spent over 8 hours sitting. Yet it is important to note that due to the cross-sectional nature of the study, it could be argued that the participants who were more depressed, anxious and had lower levels of vitality could be more prone to spending greater amounts of time sitting. Furthermore, this association was only evident during the weekdays. Contextual information regarding the type of sedentary activities participated in during measured sitting time and where sitting behavior occurred was not obtained. This is an identifiable limitation of the study, along with the cross-sectional nature of the data, making it difficult to infer cause and effect. This would have allowed us to further explore the workplace as a potential important context for sedentary behavior and mental health. Whilst our findings suggest a large positive effect for sitting less than 8 hours per day on improved mental well-being, we acknowledge a requirement for further research with larger groups of participants in order to translate our findings into meaningful recommendations. Our data indicates that reducing sedentary behavior during the working day could be important for improving employees’ mental health and quality of life. Therefore, encouraging workplace intervention studies to include mental health and quality of life outcome measures is warranted to explore the resultant effect of reducing sedentary behavior during the working day.