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The objective of this research was to investigate the effects of gradually increasing or decreasing photosynthetic photon flux density (PPFD) during cultivation compared to a constant PPFD on biomass production. Lettuce plants (Lactuca sativa L. ‘Expertise’) were grown in climate rooms in which every three days the PPFD was increased by 16 µmol m−2 s−1 (from 140 to 300 µmol m−2 s−1 from day 0 to 30), decreased (from 300 to 140 µmol m−2 s−1), or kept constant (221 µmol m−2 s−1), while the total light integral at the end of the cultivation period (30 d) was the same for all three treatments. Gradually increasing PPFD resulted in a 16 or 13% increase in total plant dry weight compared to treatments with decreasing or constant PPFD, respectively. This increase was explained by a higher light interception mainly because, in this treatment, most of the light was provided at the end of the cultivation period when the leaf area index was high. Consequently, the light use efficiency based on incident PPFD was highest when PPFD gradually increased, even though the light use efficiency based on intercepted PPFD was highest when PPFD gradually decreased during cultivation. Despite the higher shoot dry weight when PPFD gradually increased, shoot fresh weight was not significantly affected by the light treatments. This difference in response between fresh and dry weight resulted from a higher shoot dry matter content when PPFD gradually increased. Our results show that gradually increasing PPFD had a positive effect on dry weight accumulation and increased dry matter content, but did not affect the shelf life. So, although vertical farms enable growers to keep all conditions constant, some dynamic variation of conditions might be needed for optimizing the light use efficiency.