The alchemical path is sampled at a set of discrete λ values. Therefore, this integral has to be solved numerically which introduces a bias especially when there is (strong) curvature along the alchemical pathway. Since sufficient sampling in all λ states is necessary to obtain reliable free energy estimates for both estimators, enhanced sampling techniques, such as Hamiltonian Replica Exchange (HREX), are often used to improve the sampling of configuration space. Here, λ windows are run in parallel, so that the simulations can swap configurations of the system between the states. Energy barriers of the potential energy surface might be crossed more often and earlier since the energy landscape differs among the states of the alchemical pathway. The Metropolis acceptance criterion ensures that simulations in all states still converge towards the equilibrium distribution. As mentioned in the introduction, all λ windows need to be simulated at equilibrium in EQ calculations, and some of these can require sampling unphysical states which might have even slower correlation times than the physical states, necessitating very long sampling times despite the use of enhanced sampling techniques. 2.3. Non-equilibrium calculations drive rapid transitions between physical end states. In contrast, non-equilibrium FEC sample only the physical end states at equilibrium while the alchemical path is explored in fast (non-equilibrium) transitions. After generating an ensemble of equilibrium conformations in the initial and final state, MD simulations are performed which rapidly switch the ligand from interacting to non-interacting and vise versa (Figure ). By changing the parameter λ, the system is forced to quickly change to the other end state without reaching equilibrium at any intermediate state. Consequently, there is dissipated work along the path and the average work exceeds the free energy difference according to the second law of thermodynamics and the maximum work theorem