A systematic study was performed on the influence of charge and steric hindrance on the assembly into fibres of a series of pentameric peptides based on the well-known β-sheet forming sequence Gly-Ala-Gly-Ala-Gly, which were N-terminally acylated with pentacosadiynoic acid. To investigate the effect of steric hindrance and charge repulsion on the fibre structure, either the N-terminal or the C-terminal amino acid in the sequence was replaced by a glutamic acid or lysine residue. Furthermore, peptide amphiphiles (PAs) with an amide or a free acid group at the C-terminus were compared. Steric hindrance and charge repulsion were addressed individually by varying the pH during and after fibre preparation. The self-assembled structures were examined with circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM). UV spectroscopy was used to probe the diacetylene packing in the hydrophobic tail, both by polymerisation behaviour and chromatic properties of the polymers. In brief, the assembly was hindered more if the modification was close to the alkyl tail, and glutamic acid brought about a larger effect than lysine. PAs with two charges yielded assemblies which after polymerisation were found to be the most susceptible towards changes in pH, behaving as a colour-based pH sensor. Typically, TEM and UV showed the same trends, indicating that a distorted morphology as observed with TEM is indicative of a poorer molecular packing of the peptide amphiphile fibres, probed via the changes in absorption of the polydiacetylene backbone.