<p>This review describes various molecular biophysics methods (crystallography, mass spectrometry, NMR spectroscopy, electron cryo-microscopy&#xa0;(cryoEM),&#xa0;free electron laser and X-ray photon correlation spectroscopy) which can be used to investigate the molecular structure of proteins under diverse conditions (visually summarised in the thumbnail image for the journal contents pages). We focus particularly on those which permit for the incubation and/or experimental sample analysis at mammalian body temperature (37&#xa0;°C) or at physiological conditions for yet higher temperatures such as thermophiles. Crystallography, a leading method in structure elucidation, in recent decades has been dominated by structures analysed at cryogenic temperatures to ensure best resolution and crystal stability under X-ray irradiation. However, it raises the question—is the atomic structure elucidated by cryo-crystal structures truly representative of processes occurring at body temperature? This is surely an important requirement for protein–ligand binding investigations for drug discovery as protein binding may vary with temperature and indicate key aspects that could be overlooked. A review of wwPDB submissions versus sample temperature analysis clearly indicates a marked lack of atomic data obtained at 37&#xa0;°C. This is not to say that 100&#xa0;K cryo-crystallography ought to be replaced, in such structure-based drug discovery which is highly efficiently implemented at many macromolecular crystallographic beamlines worldwide, but favoured ligand binding events in particular for lead compounds from those surveys may provide additional valued data when studied at 37&#xa0;°C.</p> Graphical abstract <p>Thumbnail contents image: Molecular biophysics methods suitable for the analysis of&#xa0;macromolecules at body temperatures or higher</p> <p></p>