Laboratories that study Drosophila melanogaster or other insects commonly use carbon dioxide (CO2) anaesthesia for sorting or other work. Unfortunately, the use of CO2 has potential unwanted physiological effects, including altered respiratory and muscle physiology, which impact motor function behaviours. The effects of CO2 at different levels and exposure times were examined on the subsequent recovery of motor function as assessed by climbing and flight assays. With as little as a five minute exposure to 100% CO2, D. melanogaster exhibited climbing deficits up to 24 hours after exposure. Any exposure length over five minutes produced climbing deficits that lasted for days. Flight behaviour was also impaired following CO2 exposure. Overall, there was a positive correlation between CO2 exposure length and recovery time for both behaviours. Furthermore, exposure to as little as 65% CO2 affected the motor capability of D. melanogaster. These negative effects are due to both a CO2-specific mechanism and an anoxic effect. These results indicate a heretofore unconsidered impact of CO2 anaesthesia on subsequent behavioural tests revealing the importance of monitoring and accounting for CO2 exposure when performing physiological or behavioural studies in insects.