The production of Cannabis sp. under inductive photoperiods requires investment in lighting infrastructure to delay the inflorescence stage and maintain yield. Previous reports have shown that the interruption of the night phase (Night Break, NB) prevents floral development, however, the effects related to stress response, yield and cannabinoid concentration are unknown for cannabis. The present study sought to study the impact of NB in Cannabis sp. growth, physiology, and yield. Four photoperiod treatments were compared: long day (T1: 18 h light/6 h dark), night break treatments with 12 h light photoperiods supplemented with 1 h light pulse (T2, at ZT+17)) or with four light pulses of 15 minutes each (T3: ZT+15 to ZT+18) and short-day treatment (T4: 12h light /12h dark). The appearance of pistils and reduction of internodes in apical segments were observed after 20 days only in plants subjected to short day (T4), while all four treatments displayed a similar physiological behavior. Regarding yield, plants with supplemental light presented higher biomass accumulation than the short-day treatment. Cannabinoid accumulation profiles showed distinct trends: CBD accumulation showed no differences, while THC accumulation was lower in T3 and T4 compared to T1 and T2. These results suggest that night interruption in non-psychoactive plants is as effective as extending the light phase in preventing inflorescence formation, without generating stress or reductions in yield.