A large variety of functional self-assembled supramolecular nanostructures have been reported over recent decades.1 The experimental approach to these systems initially focused on the design of molecules for specific interactions that lead to discrete geometric structures.1–4 Recently, kinetics and mechanistic pathways of self-assembly have been investigated,6,7 but there remains a major gap in our understanding of internal conformational dynamics and their links to function. This challenge has been addressed through computational chemistry with the introduction of molecular dynamics (MD) simulations, which yield information on molecular fluctuations over time.5–7 Experimentally, it has been difficult to obtain analogous data with sub-nanometer spatial resolution. Thus, there is a need for experimental dynamics measurements, to confirm and guide computational efforts and to gain insight into the internal motion in supramolecular assemblies. Using site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy, we measured conformational dynamics through the 6.7 nm cross-section of a self-assembled nanofiber in water and provide unique insight for the design of supramolecular functional materials.