We have performed extended MD simulations using Hamiltonian Replica Exchange and standard MD on two macrocyclic hosts, compound 1 (see Figure ) and compound 2 (see Figure ) that selectively bind small guest molecules via an induced fit and a conformational selection mechanism, respectively. HREM and MD simulations have been also performed on the ODR-BRD4(I) complex in TIP3 water, with the ligand occupying two competing poses (see Figure ). In all cases, we used standard HREM based on the scaling protocol defined in Eq. 3 and an adaptive HREM with acceptance ratio balancing implemented via Eq. 4. Adaptive HREM seems to have a moderate positive impact on the average round-trip-time and on the replica flux, resulting in a marginal effect on the sampling efficiency. "One off" MD simulations, even with a total simulation time exceeding by one order of magnitude that of the HREM simulations, are unable to reliably sample the conformational space in the two macrocyclic hosts and in BDR4(I)-ODR complex. By distributing the total simulation time in a series of shorter independent standard MD replicates, the sampling efficiency is restored in the case compound 1 and in the BRD4(I)-ODR complex where conformational sub-states or secondary poses are separated by free energy barriers not exceeding 1-2 kcal/mol. For compound 2, characterized by long-lived metastable structures separated by barriers exceeding 4 kcal/mol, HREM appears to be the only viable alternative for a reliable canonical sampling. The present results shows that in complex systems with a manifold of possible conformations, a fully converged and reproducible FEP-based approach would require costly HREM simulations on each of the intermediate alchemical states. This severe sampling issue can be in part circumvented by limiting the enhanced sampling to the end-states alone (the complex with the fully coupled ligand and the receptor in the apo structure) and recovering the binding free energy via non-equilibrium fast alchemical transformations connecting these end states.