We assessed the extent of recent environmental changes on leaf morphological (stomatal density, stomatal surface, leaf mass per unit area) and physiological traits (carbon isotope composition, δ13Cleaf, and discrimination, Δ13Cleaf, oxygen isotope composition, δ18Oleaf) of two tropical rainforest species (Dicorynia guianensis; Humiria balsamifera) that are abundant in the Guiana shield (Northern Amazonia). Leaf samples were collected in different international herbariums to cover a 200 year time-period (1790–2004) and the whole Guiana shield. Using models describing carbon and oxygen isotope fractionations during photosynthesis, different scenarios of change in intercellular CO2 concentrations inside the leaf (Ci), stomatal conductance (g), and photosynthesis (A) were tested in order to understand leaf physiological response to increasing air CO2 concentrations (Ca). Our results confirmed that both species displayed physiological response to changing Ca. For both species, we observed a decrease of about 1.7‰ in δ13Cleaf since 1950, without significant change in Δ13Cleaf and leaf morphological traits. Furthermore, there was no clear change in δ18Oleaf for Humiria over this period. Our simulation approach revealed that an increase in A, rather than a decrease in g, explained the observed trends for these tropical rainforest species, allowing them to maintain a constant ratio of Ci/Ca.