Ensemble-based measurements of kinetic isotope effects (KIEs) have advanced physical understanding of enzyme-catalysed reactions, but controversies remain. KIEs are used as reporters of rate-limiting H-transfer steps, quantum mechanical tunnelling, dynamics and multiple reactive states. Single molecule enzymatic KIEs could provide new information on the physical basis of enzyme catalysis. Here single pair fluorescence energy transfer (spFRET) was used to measure single molecule (SM) enzymatic KIEs on the H-transfer catalysed by the enzyme pentaerythritol tetranitrate reductase. We evaluated a range of methods for extracting the SM KIE from single molecule spFRET time traces. The SM KIE enabled separation of contributions from non-enzymatic protein and fluorophore processes and H-transfer reactions. Our work demonstrates SM KIE analysis as a new method for deconvolving reaction chemistry from intrinsic dynamics.