Single-particle cryogenic electron microscopy (cryo-EM), a powerful class of techniques in structural biology with the potential to resolve molecular ensembles, requires that samples be vitrified to protect them from the high vacuum of the electron microscope. However, the effects of vitrification on biomolecular ensembles are unknown. To study them, we carried out extensive molecular dynamics simulations (over 50 ms) of the Trp-cage miniprotein at equilibrium and undergoing rapid cooling. We simulated seven cooling rates spanning three orders of magnitude, with the slowest coolings matching experimental rates. By inspecting molecular mobility and density-temperature trajectories for water with and without protein, we found that water vitrification is unaltered by the protein. To track protein conformation changes, and to relate them to kinetics, we made a Markov State Model (MSM) of Trp-cage from 5.4 ms of equilibrium sampling at 277 K. We observed that MSM states with a characteristic time longer than the duration of the nonequilibrium cooling tend to be more robust to artifacts induced by such cooling. Critically, although we observe that some states vanish in the equilibrium ensemble at 230 K, none do in our nonequilibrium cooled ensembles. To account for these perturbations, we developed a thermodynamic inference framework for recovering equilibrium populations from vitrified ensembles. These results indicate that cryo-EM has the capacity to be an accurate technique for the study of biomolecular ensembles.