BACKGROUND: Many large studies have implemented wrist or thigh accelerometry to capture physical activity, but the accuracy of these measurements to infer activity energy expenditure (AEE) and consequently total energy expenditure (TEE) has not been demonstrated. The purpose of this study was to assess the validity of acceleration intensity at wrist and thigh sites as estimates of AEE and TEE under free-living conditions using a gold-standard criterion. METHODS: Measurements for 193 UK adults (105 men, 88 women, aged 40-66 years, BMI 20.4-36.6 kg m-2) were collected with triaxial accelerometers worn on the dominant wrist, non-dominant wrist and thigh in free-living conditions for 9-14 days. In a subsample (50 men, 50 women) TEE was simultaneously assessed with doubly labelled water (DLW). AEE was estimated from non-dominant wrist using an established estimation model, and novel models were derived for dominant wrist and thigh in the non-DLW subsample. Agreement with both AEE and TEE from DLW was evaluated by mean bias, root mean squared error (RMSE), and Pearson correlation. RESULTS: Mean TEE and AEE derived from DLW were 11.6 (2.3) MJ day-1 and 49.8 (16.3) kJ day-1 kg-1. Dominant and non-dominant wrist acceleration were highly correlated in free-living (r = 0.93), but less so with thigh (r = 0.73 and 0.66, respectively). Estimates of AEE were 48.6 (11.8) kJ day-1 kg-1 from dominant wrist, 48.6 (12.3) from non-dominant wrist, and 46.0 (10.1) from thigh; these agreed strongly with AEE (RMSE ~12.2 kJ day-1 kg-1, r ~ 0.71) with small mean biases at the population level (~6%). Only the thigh estimate was statistically significantly different from the criterion. When combining these AEE estimates with estimated REE, agreement was stronger with the criterion (RMSE ~1.0 MJ day-1, r ~ 0.90). CONCLUSIONS: In UK adults, acceleration measured at either wrist or thigh can be used to estimate population levels of AEE and TEE in free-living conditions with high precision.