Flagellin (flg22) induces rapid and long-lasting defence responses. It may also affect the photosynthetic activity depending on several internal and external factors, such as the phytohormone ethylene or the day/night time. Based on the results, flg22 treatment, neither in the light phase nor in the evening, caused any significant change in chlorophyll fluorescence induction parameters in the leaves of wild-type and ethylene-receptor mutant Never ripe tomato plants measured the next morning. However, flg22 in the light phase decreased the effective quantum yield and the photochemical quenching both locally and systemically in guard cells. In parallel, the production of reactive oxygen species and nitric oxide increased, which contributed to the stomatal closure and a decrease in CO2 assimilation the next day. A decrease in sugar content and elevated hexokinase activity measured after flg22 exposure can also contribute to local defence responses in intact tomato plants.