Non-homogeneous mixing of methane (NHM) within closed chambers was studied under laboratory conditions. The experimental set-up consisted of a PVC vented chamber of 5.3 litres of effective volume fitted with a power-adjustable 12 V fan. NHM within the chamber was studied according to fan position (top vs lateral), fan airflow strength (23 vs 80 cubic feet per minute) and the mixing time before sample withdrawal (5, 10, 15 and 20 minutes). The potential bias of methane flux densities caused by NHM was addressed by monitoring the difference between linearly expected and estimated flux densities of ca. 400, ca. 800 and ca. 1,600 mg CH4.m−2 d−1. Methane within the chamber was under non-homogeneous conditions. Accordingly, methane concentrations at the bottom of the chamber were between 20 to 70% higher than those recorded at the middle or top sections of the chamber, regardless of fan position, fan air-flow strength or time before sample withdrawal. NHM led to notable biases on flux density estimation. Accordingly, flux density estimated from top and middle sampling sections were systematically lower (ca. 50%) than those expected. Flux densities estimated from bottom samples were between 10% higher and 25% lower than expected, regardless of the flux density considered.