OBJECTIVE: To validate questionnaire-based physical activity level (PAL) against accelerometry and a 24 h physical activity diary (24 h AD) as reference methods (Protocol 2), after validating these reference methods against the heart rate-oxygen consumption (HRVO2) method (Protocol 1). DESIGN: Cross-sectional study. SETTING: Two villages in Andhra Pradesh state and Bangalore city, South India. SUBJECTS: Ninety-four participants (fifty males, forty-four females) for Protocol 2; thirteen males for Protocol 1. RESULTS: In Protocol 2, mean PAL derived from the questionnaire (1.72 (sd 0.20)) was comparable to that from the 24 h AD (1.78 (sd 0.20)) but significantly higher than the mean PAL derived from accelerometry (1.36 (sd 0.20); P < 0.001). Mean bias of PAL from the questionnaire was larger against the accelerometer (0.36) than against the 24 h AD (-0.06), but with large limits of agreement against both. Correlations of PAL from the questionnaire with that of the accelerometer (r = 0.28; P = 0.01) and the 24 h AD (r = 0.30; P = 0.006) were modest. In Protocol 1, mean PAL from the 24 h AD (1.65 (sd 0.18)) was comparable, while that from the accelerometer (1.51 (sd 0.23)) was significantly lower (P < 0.001), than mean PAL obtained from the HRVO2 method (1.69 (sd 0.21)). CONCLUSIONS: The questionnaire showed acceptable validity with the reference methods in a group with a wide range of physical activity levels. The accelerometer underestimated PAL in comparison with the HRVO2 method.