Our understanding of human energy metabolism has benefited greatly from the application of water labelled with 2H and 18O for the measurement of total body water, water turnover and total daily energy expenditure. Applications include validation of techniques for the assessment of dietary intake and physical activity, assessment of water and energy requirement and the assessment of the effect of dietary and physical activity interventions, including its use with endurance athletes competing at the highest level. Critical aspects of the application are isotope dose preparation, sample collection, sample analysis and the calculation procedure. The labelled-water method can easily be applied in normal living conditions, including exercise, and in the clinical setting. However, sample analysis requires a sophisticated laboratory with an isotope-ratio mass spectrometer and a sample preparation system. Examples of insights based on labelled-water studies are: (1) self-reported dietary intakes often underestimate energy requirements; (2) subjects have problems maintaining energy balance when daily energy expenditure exceeds 2.5 times resting energy expenditure. Devices for the assessment of physical activity validated using labelled water allow the study of activity patterns and strategies to influence the activity level of a sedentary society.