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 implemented with a power-adjustable 12V fan. NHM was addressed by injecting a known volume of methane and extracting, afterwards, gas samples from the top, middle and bottom sections of the chamber. NHM within the chamber was studied under different experimental situations such as the 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. 1600 mg CH4.m-2.d-1. Results showed that methane within the chamber was under non-homogeneous conditions. Accordingly, for mixing times before sample withdrawal ranging from 5 to 20 minutes 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 the fan position or fan airflow strength. Furthermore, 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, regardless the flux density considered. Alternatively, flux densities estimated from bottom samples showed a lesser bias (between 10% higher to 25% lower than expected flux densities).