Background: Prolonged sitting is associated with an increased risk of chronic ill-health. Although high levels of sedentary behaviour are documented in desk-based workers, there are few data examining this in organizations with greater job diversity such as the National Health Service (NHS). Aims: To assess the association of occupational and non-occupational sedentary behaviours with key demographic and occupational characteristics of NHS workers to help inform policy development. Methods: A cross-sectional survey conducted in a large NHS teaching hospital in the north of England. Volunteer members of staff were asked to complete a web- or paper-based version of the Workforce Sitting Questionnaire. Demographic and occupational data collected included age, gender, ethnicity, educational attainment, occupational group; full- or part-time status and whether the participant reported their job as desk-based. Descriptive statistics and 95% confidence intervals were calculated to compare sedentary behaviour across demographic and job characteristics. Results: Clerical and desk-based NHS workers were more sedentary at work than other colleagues. New findings identified that NHS workers aged under 30 and those without educational qualifications had high levels of sedentary behaviour outside work. After adjusting for working hours, part-time employees were significantly more likely to be sedentary. Conclusions: Our results have implications for those developing strategies to reduce sedentary behaviour in the NHS workforce. Whilst standing desks have received much interest, alternative approaches may be attractive to NHS employers in reducing workplace and non-occupational sitting time. These may also be relevant to other organizations.