In a lab-scale bioreactor system, (20 L of effective volume in our study) controlling a constant temperature inside bioreactor with a total volume 25 L is a simple process, whereas it is a complicated process in the actual full-scale system. There might exist a localized temperature difference inside the reactor, affecting bioenergy yield. In the present work, the temperature at the middle layer of bioreactor was controlled at 35 °C, while the temperature at top and bottom of bioreactor was controlled at 35 ± 0.1, ±1.5, ±3.0, and ±5.0 °C. The H₂ yield of 1.50 mol H₂/mol hexose_added was achieved at ±0.1 and ±1.5 °C, while it dropped to 1.27 and 0.98 mol H₂/mol hexose_added at ±3.0 and ±5.0 °C, respectively, with an increased lactate production. Then, the reactor with automatic agitation speed control was operated. The agitation speed was 10 rpm (for 22 h) under small temperature difference (<±1.5 °C), while it increased to 100 rpm (for 2 h) when the temperature difference between top and bottom of reactor became larger than ±1.5 °C. Such an operation strategy helped to save 28% of energy requirement for agitation while producing a similar amount of H₂. This work contributes to facilitating the upscaling of the dark fermentation process, where appropriate agitation speed can be controlled based on the temperature difference inside the reactor.