The biological production of hydrogen from cassava starch wastewater (CSW) was evaluated in an anaerobic fixed-bed reactor (UAFBR). The assays were carried out to evaluate the effects of organic loading rate (OLR) increase and strategies of inoculation (AS anaerobic sludge thermally treated and NF - naturally fermented cassava starch wastewater) on UAFBR performance. The OLR increase (10-20 g L-1 d(-1)) associated with hydraulic retention time (HRT) decrease (4-2 h) improved the volumetric hydrogen production rate (VHPR, from 229 to 550 mLH(2).L-1.d(-1)), molar hydrogen flow rate (MHFR, from 1.0 to 2.5 mmolH(2).h(-1)) and hydrogen yield (HY, from 0.2 to 0.3 molH(2).mol(-1)Carb) from CSW due to increase in substrate availability. Both inoculation alternatives (AS and NF) were effective for the selection of acidogenic microorganisms, which demonstrates that NF could be considered a simple and economic alternative for the acquisition of inoculum for continuous acidogenic reactors. Hydrogen production decreased after 10 days of operation when the specific organic loading rate (SOLR) reached reduced values (<1 gCarb.g(-1)VSS.d(-1)), which impairs hydrogen production. For all assays, methane was present in the biogas after the 20th day of operation mainly due to biomass accumulation, which alters the biota of the reactor. Although many factors could influence the process performance in UAFBR for the production of biohydrogen, the accumulation of biomass have been pointed as the main factor in the determination of the production time, thus demanding the implementation of systematic practices to remove the excess of biomass to maintain the SOLR in levels adequate for hydrogen production. (C) 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.