Background: Breaks in prolonged sitting may have beneficial cardiometabolic and musculoskeletal health outcomes. Desk-based work settings are an important environment to promote and support breaks in sitting time. However, few studies have reported the psychometric properties of self-report measures to assess the frequency and duration of breaks from sitting. This study examined the concurrent validity and test-retest reliability of the Workplace Sitting Breaks Questionnaire (SITBRQ) designed to assess frequency and duration of breaks in sitting within desk-based work settings. Methods: To assess the concurrent validity, a sample of 147 desk-based employees completed the SITBRQ and wore an Actigraph GT1M accelerometer for seven consecutive days. To establish test-retest reliability, SITBRQ was administered on two separate occasions 7-14 days apart to a separate sample of 96 desk-based employees. Results: A low relative agreement with accelerometry (Spearman's r = 0.24 [95% CI 0.07-0.40]) was determined for self-reported frequency, but not for the duration of sitting breaks (Spearman's r = 0.05 [95% CI -0.12-0.22]). Adequate reliability was determined for both self-reported frequency (Spearman's r = 0.71 [95% CI 0.59-0.79], Cohen's kappa = 0.74 [95% CI 0.64-0.84]) and duration of sitting breaks (Spearman's r = 0.59 [95% CI 0.45-0.71], Cohen's kappa = 0.61 [95% CI 0.38-0.85]). Conclusion: SITBRQ may be used for assessment of the frequency of sitting breaks within desk-based work settings with validity and reliability similar to other self-reports in the field of sedentary behaviour research. However, until adequately improved and re-evaluated, it should not be used to collect data about the duration of breaks in sitting time.