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Sitting at work is an emerging occupational health risk. Few instruments designed for use in population-based research measure occupational sitting and standing as distinct behaviors. This study aimed to develop and validate brief measure of occupational sitting and physical activity.A convenience sample (n = 99, 61% female) was recruited from two medium-sized workplaces and by word-of-mouth in Sydney, Australia. Participants completed the newly developed Occupational Sitting and Physical Activity Questionnaire (OSPAQ) and a modified version of the MONICA Optional Study on Physical Activity Questionnaire (modified MOSPA-Q) twice, 1 wk apart. Participants also wore an ActiGraph accelerometer for the 7 d in between the test and retest. Analyses determined test–retest reliability with intraclass correlation coefficients and assessed criterion validity against accelerometers using the Spearman ρ.The test–retest intraclass correlation coefficients for occupational sitting, standing, and walking for OSPAQ ranged from 0.73 to 0.90, while that for the modified MOSPA-Q ranged from 0.54 to 0.89. Comparison of sitting measures with accelerometers showed higher Spearman correlations for the OSPAQ (r = 0.65) than for the modified MOSPA-Q (r = 0.52). Criterion validity correlations for occupational standing and walking measures were comparable for both instruments with accelerometers (standing: r = 0.49; walking: r = 0.27–0.29).The OSPAQ has excellent test–retest reliability and moderate validity for estimating time spent sitting and standing at work and is comparable to existing occupational physical activity measures for assessing time spent walking at work. The OSPAQ brief instrument measures sitting and standing at work as distinct behaviors and would be especially suitable in national health surveys, prospective cohort studies, and other studies that are limited by space constraints for questionnaire items.