Significance The microtubule-binding protein tau misfolds into filamentous aggregates in many neurodegenerative diseases. Understanding the structure and dynamics of tau fibrils is important for designing anti-tau inhibitors. Patient-brain-derived tau fibrils have been shown to adopt disease-specific molecular conformations, but in vitro heparin-fibrillized tau was recently proposed to exhibit significant structural polymorphism. Using solid-state NMR, we show that the β-sheet core of heparin-fibrillized 0N4R tau adopts a single molecular conformation that encompasses the second and third microtubule-binding repeats, and both cysteine residues in the protein are involved in side-chain packing with other β-sheet residues. Extensive characterization of the protein dynamics indicates that these tau fibrils are heterogeneously dynamic, and the first and fourth microtubule-binding repeats are semirigid while retaining β-sheet character.