The aim of this work is to study the influence of pulsation on the start-up of lab-scale UASB reactors. Pulsation was produced by an Elastic Membrane Pulsator (EMP). The application of this device in previous works improved the performance of continuous fixed-bed fermentors and reduced the formation of preferential pathways, the retention of gas metabolites within the bed and the resistance to mass transfer. These characteristics seem to be suitable for feeding UASB reactors. In this work, the influence of pulsation frequency was studied in two pulsed UASB reactors operated in parallel with a non-pulsed one. One of them (P1) operated at high frequencies (periods of 50 and 200 s between each pulsation) and the other (P2) at low frequencies (periods of 3600 and 900 s between each pulsation). An important improvement of the removal efficiency for pulsed reactors with respect to the non-pulsed one was obtained. The structure of the biomass was observed at the end of the process by scanning electron microscopy. In general, granulation of biomass was improved when operating in pulsing form.