Whilst the importance of mixing in anaerobic digesters to enhance process performance and gas production is well recognised, the specific effects of mixing regime on biogas production are not clear. Here, the velocity gradient is used to demonstrate the importance of minimally mixed zones in a digester, with computational fluid dynamics (CFD) models indicating that 20–85% of a laboratory-scale digester experiences local velocity gradients of less than 10 s−1, dependent on mixing speed. Experimental results indicate that there is a threshold above which increased mixing speed (and hence velocity gradient) becomes counter-productive and biogas production falls. The effects of minimal mixing on digester microbiology are considered with the creation or destruction of localised pockets of high acetate concentration providing a possible explanation for the velocity gradient threshold. The identification of this threshold represents a valuable contribution to the understanding of the effects of mixing on gas production in anaerobic digesters.