Frequent breaks in prolonged sitting are associated beneficially with glycaemic control. However, the contribution of energy expenditure to this relationship has not been well characterised. In this exploratory analysis, data from 3 laboratory trials that standardised test meals, cohort characteristics (overweight/obese, sedentary), and break frequency and duration were pooled. Higher energy expenditures of different types of breaks (standing, light- or moderate-intensity walking) were associated with lower postprandial glucose and insulin responses in a dose-dependent manner.