This study evaluated the influence of the applied volumetric organic load on biohydrogen production in an anaerobic sequencing batch biofilm reactor (AnSBBR) with 3.5 L of liquid medium and treating 1.5 L of glycerin-based wastewater per cycle at 30 degrees C. Six applied volumetric organic loads (AVOL(CT)) were generated from the combination of cycle periods (3 and 4 h) and influent concentrations (3000, 4000, and 5000 mg chemical oxygen demand(COD) L-1), with values ranging from 7565 to 16,216 mg COD L-1 day(-1). No clear relationship was found between the applied volumetric organic load and the hydrogen production. However, the highest hydrogen molar production (MPr 67.5 mol H(2)m(-3) day(-1)) was reached when the reactor was operated with a cycle period of 4 h and an influent concentration of 5000 mg COD L-1 (AVOL(CT) 12,911 mg COD L-1 day(-1)). This condition also reached the highest molar yield per applied load based on the organic matter (MYAL(C,m) 21.1 mol H(2)kg COD-1). In addition, the pretreatment of the sludge/inoculum was found to not influence the productivity/yield of the process, and the use of crude glycerol as a sole source of carbon exhibited a clear disadvantage for hydrogen production compared to pure glycerol. The AnSBBR used for the hydrogen production experiments operated with pure glycerol as a sole carbon source exhibited important practical potential.