Glycerolipid remodeling, a dynamic mechanism for plant subsistence under cold stress, has been posited to affect the biophysical properties of cell membranes. In barley roots, remodeling has been observed to take place upon exposure to chilling stress and to be partially reverted during stress relief. In this study, we explored the biophysical characteristics of membranes formed with lipids extracted from barley roots subjected to chilling stress, or during a subsequent short- or long-term recovery. Our aim was to determine to what extent barley roots were able to offset the adverse effects of temperature on their cell membranes. For this purpose, we analyzed the response of the probe Laurdan inserted in bilayers of different extracts, the zeta potential of liposomes, and the behavior of Langmuir monolayers upon compression. We found important changes in the order of water molecules, which is in agreement with the changes in the unsaturation index of lipids due to remodeling. Regarding Langmuir monolayers, we found that films from all the extracts showed a reorganization at a surface pressure that depends on temperature. This reorganization occurred with an increase in entropy for extracts from control plants and without entropy changes for extracts from acclimated plants. In summary, some membrane properties were recovered after the stress, while others were not, suggesting that the membrane biophysical properties play a role in the mechanism of plant acclimation to chilling. These findings contribute to our understanding of the impact of lipid remodeling on biophysical modifications in plant roots.