This study was conducted to determine the optimal temperature difference in day–night indoor cultivation conditions to enhance the flower yield and functional component contents of female hemp plants. Hemp clones were cultivated under five distinct day and night temperature differences (DIF) during the reproductive stage. The daytime and nighttime temperature settings were as follows: 18:30 °C (negative 12 DIF), 21:27 °C (negative 6 DIF), 24:24 °C (0 DIF), 27:21 °C (positive 6 DIF), and 30:18 °C (positive 12 DIF). Seven weeks after transplantation, the growth parameters, leaf gas exchange, total phenolic compounds, 2,2-diphenylpicrylhydrazyl scavenging activity, and cannabinoid contents were analyzed. The total shoot biomass based on dry weight was highest at 21:27, reaching 41.76 g, and lowest at 30:18, measuring 24.46 g. However, the flower biomass, which is the primary production site, was highest at 24:24 and lowest at 18:30, showing a 4.7-fold difference. The photosynthesis-related parameters were temperature-dependent and strongly correlated with biomass production. The cannabinoid content of the hemp leaves increased at 21:27, whereas that of the hemp flowers increased at 27:21. The findings of this study indicate that the optimal temperature condition for female hemp flower production in a limited space is positive 6 DIF treatment, which corresponds to 27:21 °C. These results can contribute to advancements in indoor crop cultivation technology.
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