A survey of tree crown defoliation and leaf physiological traits (chlorophyll a fluorescence, nitrogen content, and stable carbon isotope composition) was carried out in the thermophilous deciduous forests in Tuscany (central Italy). In contrast to large scale surveys, where variation in defoliation can be associated with the change in environmental conditions, in a limited homogenous area the defoliation of co-existing tree species may have different significance and depends on the interaction between the characteristics of each individual species with biotic stress and environmental conditions. The survey included measurements of structural and vegetational characteristics of the forest stands, such as Leaf Area Index (LAI), basal area and tree diversity, which is expressed as the Shannon diversity index. The five tree species studied (Castanea sativa, Quercus corns, Quercus ilex, Quercus petraea and Ostrya carpinifolia) showed species-specific crown conditions and physiological features relative to stand structure and diversity. The shape of the crowns and their area (LAI) affected forest defoliation. Tree diversity reduced defoliation in C. sativa, which was the tree species most affected by defoliation, and likewise for Q. ilex. Chlorophyll a fluorescence parameters showed lower photosynthetic efficiency in defoliated C. sativa, O. carpinifolia and Q. petraea trees. Similarly, foliar nitrogen content decreased in defoliated C. sativa and O. carpinifolia trees, whereas delta C-13 was higher in defoliated C. sativa. These responses may be related to the health status of C. sativa, since it was subjected to pathogen damages and insect attacks. In contrast, the mast year in O. carpinifolia may have diverted the nutrient resources from leaves to fruits, and consequently explaining the physiological effects on the tree crown. These results suggest that the combined analysis of defoliation with foliar features and stand characteristics can provide insights into tree health and vitality.