The present work evaluated the biohydrogen production from a 0.4 L upflow anaerobic sludge blanket reactor type (UASB) operating at psychrophilic temperature (21 ± 2 °C) at different feeding strategies varying hydraulic retention times (HRT) and sucrose concentration in the feeding. First strategy (24 h/31c) fed semi-continuously 31 gsucrose L−1 at 24 h HRT; second strategy (12 h/19c) fed semi-continuously 19 gsucrose L−1 at 12 h HRT; third strategy (4 h/8c) fed continuously 8.3 gsucrose L−1 at 4 h HRT. After 70 days of operation, the UASB accumulated 65.44 L H2. The average HY for the whole operation during the three strategies was 62.6 NmL H2 gsucrose−1, and average hydrogen content was 69.04%. In general terms, the best operation strategy was 12 h/19c since it presented good set of results, the best HY (70.6 NmL H2 gsucrose−1) and a comparable hydrogen production rate (2.6 L (L d)−1) to that obtained in 4 h/8c strategy (3.17 L (L d)−1). The average gross energy potential rate from the 12 h/19c strategy was 46.21 kJ (L d)−1, whereas energy heating losses were circumvented due to operation at psychrophilic regime. Indeed, psychrophilic or room temperatures should be broadly regarded as an effective alternative towards net energy gains in biohydrogen production.