Intrinsic thermodynamic fluctuations within biomolecules are crucial for their function, and flexibility is one of the strategies that evolution has developed to adapt to extreme environments. In this regard, pressure perturbation is an important tool for mechanistically exploring the causes and effects of volume fluctuations in biomolecules and biomolecular assemblies, their role in biomolecular interactions and reactions, and how they are affected by the solvent properties. High hydrostatic pressure is also a key parameter in the context of deep-sea and subsurface biology and the study of the origin and physical limits of life. We discuss the role of pressure-axis experiments in revealing intrinsic structural fluctuations as well as high-energy conformational substates of proteins and other biomolecular systems that are important for their function and provide some illustrative examples. We show that the structural and dynamic information obtained from such pressure-axis studies improves our understanding of biomolecular function, disease, biological evolution, and adaptation.