Some systems did not select for hydrogen-producing microorganisms and an unexpected growth of hydrogenotrophic methanogens was observed, although the reactors were operated under well-defined operating conditions that could result in biohydrogen production. The aim of this study was to evaluate the effect of the organic loading rate (OLR) on the hydrogen and methane composition of the biogas produced in dark fermentative processes. The study was carried out using an upflow anaerobic sludge blanket (UASB) reactor in order to evaluate the OLR effect in systems with sludge retention. During continuous operation, the UASB reactor showed the slow development of methanogenic activity, related to the applied OLR. The results demonstrate that operating an UASB reactor at pH 5.5 is not enough to prevent the acclimation of methanogens to the acidic pH and therefore long-term biohydrogen production cannot be achieved. Moreover, this study demonstrates that OLR also has an effect on the biogas composition, where the higher the OLR the greater the biogas H2 content.