Objective: To study the relation between neck pain and work related neck flexion, neck rotation, and sitting. Methods: A prospective cohort study was performed with a follow up of 3 years among 1334 workers from 34 companies. Work related physical load was assessed by analysing objectively measured exposure data (video recordings) of neck flexion, neck rotation, and sitting posture. Neck pain was assessed by a questionnaire. Adjustments were made for various physical factors that were related or not related to work, psychosocial factors, and individual characteristics. Results: A significant positive relation was found between the percentage of the working time in a sitting position and neck pain, implying an increased risk of neck pain for workers who were sitting for more than 95% of the working time (crude relative risk (RR) 2.01, 95% confidence interval (95% CI) 1.04 to 3.88; adjusted RR 2.34, 95% CI 1.05 to 5.21). A trend for a positive relation between neck flexion and neck pain was found, suggesting an increased risk of neck pain for people working with the neck at a minimum of 20° of flexion for more than 70% of the working time (crude RR 2.01, 95% CI 0.98 to 4.11; adjusted RR 1.63, 95% CI 0.70 to 3.82). No clear relation was found between neck rotation and neck pain. Conclusion: Sitting at work for more than 95% of the working time seems to be a risk factor for neck pain and there is a trend for a positive relation between neck flexion and neck pain. No clear relation was found between neck rotation and neck pain.