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Due to the prognosticated warming in late autumn in northern and central Europe, the objective of this investigation was to establish whether temperature increase under low radiation conditions affect the content of phytochemicals such as carotenoids, chlorophylls and glucosinolates, and the also antioxidative effective ascorbic acid in broccoli. Greenhouse grown broccoli, cv. Marathon, was cultivated after head induction at three different daily mean temperatures (in the range from 7.2 to 19.7
°C) under two different daily mean radiation levels (in the range from 1.9 to 13.4
mol
m
−2
day
−1). Contents of ascorbic acid, lutein and alkyl glucosinolates (especially glucoraphanin) increased at daily mean temperatures between 7–13
°C combined with a moderate daily mean radiation of 10–13
mol
m
−2
day
−1. The alkyl glucosinolates glucoraphanin and glucoiberin increased 8-fold and ascorbic acid 0.5-fold, whereas the main indole glucosinolate glucobrassicin was reduced. The data provided here have implications for quality-oriented production and crop management strategies in warmed up autumn periods aiming to optimize health-promoting substance content in broccoli under low radiation conditions.