Respiratory chambers are the current gold standard for assessing human energy expenditure and substrate utilization over a long period of time (several hours to several days), based on oxygen consumption, carbon dioxide production, and urinary nitrogen excretion. Analysis of human energy metabolism using a respiratory chamber provides information about the total energy expenditure (TEE), sleeping metabolic rate (SMR), resting metabolic rate, dietinduced thermogenesis (DIT), activity-induced thermogenesis (AIT), and substrate oxidation. In this review, we describe the theoretical underpinnings of the respiratory chamber, as well as the measurement reproducibility and applications as study endpoints for indirect calorimetry. In humans, the coefficients of variation in energy expenditure and substrate utilization were estimated by 24-h repeatability studies. Under the appropriate conditions, the coefficients of variation for TEE were 1% to 5%, SMR was around 1%, DIT was around 40%, AIT was around 10%, and substrate oxidation was around 5%. Factors that impact energy expenditure and substrate oxidation have been reported, and future weight changes can be predicted based on the 24-h respiratory quotient and substrate oxidation. As the 24-h energy expenditure and substrate oxidation are affected by the 24-h energy balance, it is important to consider the subject's energy balance prior to and during calorimetry. Accurate measurements of energy and substrate balance (intake minus utilization) will contribute to a better understanding of the conditions that lead to changes in body weight. Properly obtaining measurements using a respiratory chamber requires a thorough understanding of the measurement principles and calculation methods, as well as an appropriate protocol.