The enthalpic and entropic components of ligand–protein binding free energy reflect the interactions and dynamics between ligand and protein. Despite decades of study, our understanding and hence our ability to predict these individual components remains poor. In recent years, there has been substantial effort and success in the prediction of relative and absolute binding free energies, but the prediction of the enthalpic (and entropic) contributions in biomolecular systems remains challenging. Indeed, it is not even clear what kind of performance in terms of accuracy could currently be obtained for such systems. It is, however, relatively straight-forward to compute the enthalpy of binding. We thus evaluated the performance of absolute enthalpy of binding calculations using molecular dynamics simulation for ten inhibitors against a member of the bromodomain family, BRD4-1, against isothermal titration calorimetry data. Initial calculations, with the AMBER force-field showed good agreement with experiment (R2= 0.60) and surprisingly good accuracy with an average of root-mean-square error (RMSE) = 2.49 kcal mol−1. Of the ten predictions, three were obvious outliers that were all over-predicted compared to experiment. Analysis of various simulation factors, including parameterization, buffer concentration and conformational dynamics, revealed that the behaviour of a loop (the ZA loop on the periphery of the binding site) strongly dictates the enthalpic prediction. Consistent with previous observations, the loop exists in two distinct conformational states and by considering one or the other or both states, the prediction for the three outliers can be improved dramatically to the point where theR2= 0.95 and the accuracy in terms of RMSE improves to 0.90 kcal mol−1. However, performance across force-fields is not consistent: if OPLS and CHARMM are used, different outliers are observed and the correlation with the ZA loop behaviour is not recapitulated, likely reflecting parameterization as a confounding problem. The results provide a benchmark standard for future study and comparison.