No current validated survey instrument allows a comprehensive assessment of both physical activity and travel behaviours for use in interdisciplinary research on walking and cycling. This study reports on the test-retest reliability and validity of physical activity measures in the transport and physical activity questionnaire (TPAQ).The TPAQ assesses time spent in different domains of physical activity and using different modes of transport for five journey purposes. Test-retest reliability of eight physical activity summary variables was assessed using intra-class correlation coefficients (ICC) and Kappa scores for continuous and categorical variables respectively. In a separate study, the validity of three survey-reported physical activity summary variables was assessed by computing Spearman correlation coefficients using accelerometer-derived reference measures. The Bland-Altman technique was used to determine the absolute validity of survey-reported time spent in moderate-to-vigorous physical activity (MVPA).In the reliability study, ICC for time spent in different domains of physical activity ranged from fair to substantial for walking for transport (ICC = 0.59), cycling for transport (ICC = 0.61), walking for recreation (ICC = 0.48), cycling for recreation (ICC = 0.35), moderate leisure-time physical activity (ICC = 0.47), vigorous leisure-time physical activity (ICC = 0.63), and total physical activity (ICC = 0.56). The proportion of participants estimated to meet physical activity guidelines showed acceptable reliability (k = 0.60). In the validity study, comparison of survey-reported and accelerometer-derived time spent in physical activity showed strong agreement for vigorous physical activity (r = 0.72, p<0.001), fair but non-significant agreement for moderate physical activity (r = 0.24, p = 0.09) and fair agreement for MVPA (r = 0.27, p = 0.05). Bland-Altman analysis showed a mean overestimation of MVPA of 87.6 min/week (p = 0.02) (95% limits of agreement -447.1 to +622.3 min/week).The TPAQ provides a more comprehensive assessment of physical activity and travel behaviours and may be suitable for wider use. Its physical activity summary measures have comparable reliability and validity to those of similar existing questionnaires.