Kale (Brassica oleracea var acephala) is known as a vegetable with good tolerance of environmental stress and numerous beneficial properties for human health, which are attributed to different phytochemicals. In the present study, investigation of how low temperatures affect proline, pigments and specialized metabolites content was performed using 8-weeks old kale plants subjected to chilling (at 8 °C, for 24 h) followed by short freezing (at −8 °C, for 1 h after previous acclimation at 8 °C, for 23 h). Plants growing at 21 °C served as a control. In both groups of plants (exposed to low temperatures and exposed to short freezing) a significant increase in proline content (14% and 49%, respectively) was recorded. Low temperatures (8 °C) induced an increase of pigments (total chlorophylls 7%) and phytochemicals (phenolic acids 3%; flavonoids 5%; carotenoids 15%; glucosinolates 21%) content, while exposure to freezing showed a different trend dependent upon observed parameter. After freezing, the content of chlorophylls, carotenoids, and total phenolic acids retained similar levels as in control plants and amounted to 14.65 ± 0.36 mg dw g−1, 2.58 ± 0.05 mg dw g−1 and 13.75 ± 0.07 mg dw CEA g−1, respectively. At the freezing temperature, total polyphenol content increased 13% and total flavonoids and glucosinolates content decreased 21% and 54%, respectively. Our results suggest that acclimatization (23 h at 8 °C) of kale plants can be beneficial for the accumulation of pigments and phytochemicals, while freezing temperatures affect differently specialized metabolite synthesis. The study suggests that growing temperature during kale cultivation must be considered as an important parameter for producers that are orientated towards production of crops with an increasing content of health-related compounds.