- Designed amphiphilic β-sheet peptides with the sequence Pro−Glu−(Phe−Glu)n−Pro (n = 2−7) were previously shown by grazing incidence X-ray diffraction (GIXD), to form ordered two-dimensional (2-D) monolayer structures at interfaces induced by the proline residues at peptide termini. The GIXD diffraction pattern was modeled with two coexisting lattice arrangements, suggesting structural flexibility exhibited in the multiple ways by which β-strands and their amino acid side chains pack into ordered 2-D structures. Here, we find by in-situ GIXD measurements that the ordered β-sheet assemblies may undergo a quasi-reversible compression and expansion cycle at the air−water interface. The diffraction measurements indicate that on compression the repeat distance that corresponds to the long axes of the peptide strands may decrease by up to 37% in length. Upon expansion the compressed β-sheet assemblies revert elastically to their original conformation. The interstrand repeat distance along the peptide hydrogen bonds apparently does not change along the film compression and expansion. Based on the GIXD data, at surface pressures higher than ∼3 mN/m, beyond the peptide limiting area per molecule, the compressibility is 7.4 ± 0.6 m/N. The out-of-plane Bragg rod diffraction patterns imply that in the compressed state the β-strands buckle up in reaction to the increase in surface pressure. At low surface pressure, the 2-D compressibility of the crystalline β-sheet was estimated at ∼32 m/N attributed to interdomain rearrangements.