Conductance of CO2 across the mesophyll (Gm) frequently constrains photosynthesis (PN) but cannot be measured directly. We examined Gm of cherry (Prunus avium L.) subjected to severe drought using the variable J method and carbon-isotopic composition (δ13C) of sugars from the centre of the leaf, the leaf petiole sap, and sap from the largest branch. Depending upon the location of the plant from which sugars are sampled, Gm may be estimated over scales ranging from a portion of the leaf to a canopy of leaves. Both the variable J and δ13C of sugars methods showed a reduction in Gm as soil water availability declined. The δ13C of sugars further from the source of their synthesis within the leaf did not correspond as closely to the diffusive and C-isotopic discrimination conditions reflected in the instantaneous measurement of gas exchange and chlorophyll-fluorescence utilised by the variable J approach. Post-photosynthetic fractionation processes and/or the release of sugars from stored carbohydrates (previously fixed under different environmental and C-isotopic discrimination conditions) may reduce the efficacy of the δ13C of sugars from leaf petiole and branch sap in estimating Gm in a short-term study. Consideration should be given to the spatial and temporal scales at which Gm is under observation in any experimental analysis.