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Flow-through respirometry systems that measure oxygen consumption (VO2), carbon dioxide production (VCO2) and evaporative water loss (EWL) require the accurate calibration of the flow meter and three separate analysers (O2, CO2 and H2O vapour). Correct measurement of VO2, VCO2 and EWL depends on the incurrent air flow (VI) and its condition (e.g. dry, CO2-free), and the excurrent air flow (VE) and its condition (e.g. dry, CO2-free), which can differ in different parts of the excurrent circuit. Usually either VI or VE is measured and the other is calculated from the gas composition. I describe here a procedure for precise calibration of CO2 and H2O analysers in a flow-through respirometry system by reference to a calibrated O2 analyser, using a small gas flame. Generic equations are derived for calculation of VO2, VO2 and EWL with a variety of configurations for a flow-through respirometry system …