Background: Dysphagia may occur in up to 44% of patients with head and neck cancer (HNC) treated with radiation therapy and up to 84% of patients treated with surgery. To test the extent of dysphagia, the 100 mL water swallow test (WST) was developed. In this study, reliability of the 100 mL WST was determined in patients with HNC and healthy subjects. Methods: Thirty-three patients and 40 healthy subjects performed the WST twice on the same day. To assess reliability, the intraclass correlation coefficient (ICC2,1), standard error of measurement, smallest detectable change, and limits of agreement were calculated. Results: Good to excellent correlations were found for patients with HNC (number of swallows; ICC = 0.923, duration; ICC = 0.893), and excellent correlations for healthy subjects (number of swallows; ICC = 0.950, duration; ICC = 0.916). Conclusion: The 100 mL WST has a good to excellent reliability in patients with HNC and healthy subjects.