Immobilization of insects is necessary for various experimental purposes, and CO2 exposure remains the most popular anaesthetic method in entomological research. A number of negative side effects of CO2 anaesthesia have been reported, but CO2 probably brings about metabolic modifications that are poorly known. In this work, we used GC/MS-based metabolic fingerprinting to assess the effect of CO2 anaesthesia in Drosophila melanogaster adults. We analysed metabolic variation of flies submitted to acute CO2 exposure and assessed the temporal metabolic changes during short- and long- termrecovery.We found that D.melanogaster metabotypes were significantly affected by the anaesthetic treatment. Metabolic changes caused by acute CO2 exposure were still manifested after 14 h of recovery. However, we found no evidence of metabolic alterations when a long recovery period was allowed (more than 24 h). This study points to some metabolic pathways altered during CO2 anaesthesia (e.g. energetic metabolism). Evidence of short-term metabolic changes indicates that CO2 anaesthesia should be used with utmost caution in physiological studies when a short recovery is allowed. In spite of this, CO2 treatment seems to be an acceptable anaesthetic method provided that a long recovery period is allowed (more than 24 h).