Chlorophyll fluorescence (ChF) emission is a direct indicator of the photosynthetic activity of vegetation, which is a key parameter of the carbon cycle. This paper analyses chlorophyll fluorescence evolution at leaf level during a complete diurnal cycle in simulated and natural conditions, for two species under different stress conditions. Absolute spectral radiance of the ChF emission is obtained allowing a quantitative derivation of the fluorescence yield of the ChF, which correlates well with established fluorescence instruments. The studied cases show that the ChF emission is mainly driven by the photosynthetic active radiation during the whole cycle, but the fluorescence yield is severely reduced during the central hours of the day when the plant is under stress due to light and heat. Results show that the Fraunhofer Line Discriminator method is an accurate way of retrieving quantitative values of ChF from remote sensing sensors at 760 nm and suggest that the mid‐morning period is the best time of the day to maximize signal levels while identifying vegetation stress state.