This study focused on continuous-flow hydrogen production by Thermotoga neapolitana at a hydraulic retention time (HRT) decreasing from 24 to 5 h. At each HRT reduction, the hydrogen yield (HY) immediately dropped, but recovered during prolonged cultivation at constant HRT. The final HY in each operating period decreased from 3.4 (±0.1) to 2.0 (±0.0) mol H/mol glucose when reducing the HRT from 24 to 7 h. Simultaneously, the hydrogen production rate (HPR) and the liquid phase hydrogen concentration (H) increased from 82 (±1) to 192 (±4) mL/L/h and from 9.1 (±0.3) to 15.6 (±0.7) mL/L, respectively. Additionally, the effluent glucose concentration increased from 2.1 (±0.1) to above 10 mM. Recirculating H-rich biogas prevented the supersaturation of H reaching a value of 9.3 (±0.7) mL/L, resulting in complete glucose consumption and the highest HPR of 277 mL/L/h at an HRT of 5 h.