Compared with thermodynamically equilibrium supramolecular assemblies, non-equilibrium assemblies from the same building blocks have attracted increasing attentions because their diverse structures and dynamic natures may impart the assemblies with novel functionalities. However, facile access to non-equilibrium assemblies remains a formidable challenge. Herein, we endeavored to exploit various solvent-anti-solvent methods to achieve it using peptide amphiphile C16-VVAAEE-NH₂ as a model. Through systematical utilization of dialysis, ultrasonic and stirring-dropping methods, as well as tuning of processing parameters, we demonstrated the successful formation of diverse non-equilibrium nanostructures with distinct morphologies and structures that significantly deviate from the thermodynamically favored twisted long ribbons. Additionally, these metastable nanostructures ultimately underwent spontaneous transformation into thermodynamically stable states. The transformation processes of three representative non-equilibrium assemblies were also demonstrated and characterized in detail using transmission electron microscopy, circular dichroism spectrum, and thioflavin T fluorescence spectrum. Furthermore, non-equilibrium assemblies exhibited various degrees of cytotoxic effects, which may stem from their spontaneous, dynamic transformation and interactions with cellular membranes. This study offers valuable approaches for direct access to diverse non-equilibrium supramolecular nanostructures from self-assembling peptide, and also has implications for the development of advanced materials with unprecedented biological functions.