Trees of the sweet cherry cultivar ‘Grace Star’ (Prunus avium L.) were exposed to low temperatures without frost for two consecutive nights under natural conditions 36 d after flowering, to study the effects on the physiological properties and metabolic status of leaves. The response was studied by measuring chlorophyll fluorescence and gas exchange parameters and by analyzing chloroplast pigments (i) immediately after exposure, (ii) 24 h and (iii) 48 h later. The first exposure at 2.4 (±0.2) °C and a minimum of 0.8 °C elicited more changes than the second exposure at 4.9 (±0.3) °C and a minimum of 2.4 °C. After the first exposure, the maximum quantum yield of PS II (Fv/Fm), effective quantum efficiency of PS II, net photosynthesis (PN), stomatal conductance (gs), transpiration, and intercellular CO2 concentration were significantly lower, and after the second exposure, the content of chlorophyll b, total chlorophyll, β-carotene, and lutein were lower. The content of antheraxanthin and zeaxanthin was higher immediately after both exposures, and that of antheraxanthin was also higher 24 h later. Recovery took longer in trees that were exposed twice. Fv/Fm recovered within 48 h, but the de-epoxidation state of the xanthophyll cycle pool, PN, and gs did not reach the level of controls, indicating that the stress effect lasted several days which is probably sufficient to cause fruit drop and reduce yield.