In the present study, comparison of dark fermentation (DF) efficiency from cheese whey (CW) in an anaerobic UpFlow Column Reactor (AUFCR), filled with a support material for biomass attachment and a Continuous Stirred Tank reactor (CSTR), was assessed. The process efficiency and stability as well as the effect of the operational parameters, such as the initial carbohydrates concentration (dilution factor) and the hydraulic retention time (HRT) were assessed. The experiments showed that DF of CW in a CSTR led to higher hydrogen production yields compared to the AUFCR, which was 0.36 mol of H2 per mol of consumed carbohydrates at the HRT of 12 h with initial carbohydrates concentration of 30 g/L, respectively.