Accurate measurement of energy expenditure (EE) using doubly labeled water depends on the estimate of total body water (TBW). The aims of this study were to 1) assess the accuracy of a new approach for estimating TBW and EE during high-energy turnover and 2) assess the accuracy of day-to-day assessment of EE with this new approach. EE was measured in six healthy subjects (three male) for 5 consecutive days using three doubly labeled water methods: 1) the plateau, 2) slope-intercept, and 3) overnight-slope method, with whole-room indirect calorimetry as reference method. Urine samples were collected every evening and morning. High EE (physical activity level of >2.5) was achieved by cycling 4 h·d-1. Physical activity level was 2.8 ± 0.1. TBW values were 41.9 ± 6.1, 38.4 ± 5.7, and 40.4 ± 5.8 L for the plateau, slope-intercept, and overnight-slope methods, respectively. The overnight-slope method showed the highest accuracy in estimated CO2 production, when compared with indirect calorimetry over the complete 5-d period (mean ± SD difference, 0.9% ± 1.6%). The plateau method significantly overestimated CO2 production by 4.7% ± 2.6%, whereas the slope-intercept method underestimated CO2 production (-3.4% ± 2.3%). When CO2 production was assessed per day, the overnight-slope method showed an average difference of 9.4% ± 4.5% to indirect calorimetry. The overnight-slope method resulted in a more accurate estimation of CO2 production and EE compared with the plateau or slope-intercept method over a 5-d period in high physical activity conditions. Day-to-day determination of EE using the overnight-slope method was more accurate than diet recall and several standard prediction equations in athletes.