The aim of this work was to evaluate the response of a vinasse mesophilic anaerobic digester to intermittent mixing. Physicochemical analysis were performed on the digestate, and on three samples collected at three different depths, providing a local insight of the digester before and after mixing. The mixing speed varied from 20 to 60 rpm. The biogas production gradually increased with a maximum production of 477 L CH4 ⋅kg COD − 1 at 60 rpm. Anaerobic digestion was more destabilized at higher mixing speeds, with the increase of biogas production variations. Variations in VFA (volatile fatty acids) and ammonium concentrations were apparent in parallel of biogas production variations. The optimal range of VFA concentration was 8 to 10 g⋅L − 1. The VFA / Alk ratio stabilized at the ratio of 0.5 to 0.7. The acidic zones were located in the sludge. A release of VFA from the sludge to the liquid phase was noticed after mixing.