Background: Treatment of patients with head and neck cancer can result in disrupted mastication. To measure masticatory performance in people with compromised mastication, the mixing ability test (MAT) was developed. Objective: In this study, the reliability of the MAT was evaluated in patients with head and neck cancer and healthy controls. Methods: Thirty-four patients with head and neck cancer and 42 healthy controls performed the MAT twice on the same day. To assess reliability, the intra-class correlation coefficient (ICC2,1), standard error of measurement (SEM), smallest detectable change (SDC) and limits of agreement (LoA) were calculated. Results: A good (ICC = 0.886) and moderate correlation (ICC = 0.525) were found for patients and healthy controls, respectively. Patients had a worse mixing ability (mean = 19.12, SD = 4.56) in comparison with healthy controls (mean = 16.42, SD = 2.04). The SEM was 0.76 in patients and 1.45 in healthy controls, with a SDC of 2.12 and 4.02, respectively. The LoA was −4.46 to 4.42 in patients and −3.65 to 4.59 in healthy controls. Conclusion: The MAT has a good reliability in patients with head and neck cancer and a moderate reliability in healthy controls.