Abstract Picea abies (L.) Karst. and Fagus sylvatica (L.) are important tree species in Europe, and the foreseen increase in temperature and vapour pressure deficit (VPD) could increase the vulnerability of these species. However, their physiological performance under climate change at temperate and productive sites is not yet fully understood, especially in uneven-aged stands. Therefore, we investigated tree-ring width and stable isotope chronologies (δ13C/δ18O) of these two species at 10 sites along a climate gradient in Central Europe. In these uneven-aged stands, we compared the year-to-year variability of dominant and suppressed trees for the last 80 years in relation to the sites’ spatial distribution and climate. δ18O and δ13C were generally consistent across sites and species, showing high sensitivity to summer VPD, whereas climate correlations with radial growth varied much more and depended on mean local climate. We found no significant differences between dominant and suppressed trees in the response of stable isotope ratios to climate variability, especially within the annual high-frequency signals. In addition, we observed a strikingly high coherence of the high-frequency δ18O variations across long distances with significant correlations above 1500 km, whereas the spatial agreement of δ13C variations was weaker (~700 km). We applied a dual-isotope approach that is based on known theoretical understanding of isotope fractionations to translate the observed changes into physiological components, mainly photosynthetic assimilation rate and stomatal conductance. When separating the chronologies in two time windows and investigating the shifts in isotopes ratios, a significant enrichment of either or both isotope ratios over the last decades can be observed. These results, translated by the dual-isotope approach, indicate a general climate-driven decrease in stomatal conductance. This improved understanding of the physiological mechanisms controlling the short-term variation of the isotopic signature will help to define the performance of these tree species under future climate.