Abstract Batch culture fermentations of compositionally similar but structurally distinct substrates, including resistant starches and cell wall preparations have been used to screen how substrate structure and composition affect short chain fatty acid (SCFA) production and titratable acidity. Titratable acidity was monitored using strong alkali continuously infused to prevent a pH drop below 6.5. Net acidogenic fermentation, most active during the initial 6 h, was accounted for as mainly SCFA. Differences between substrates illustrated the importance of structure in controlling fermentation yield, e.g. amylose gel residue >90% fermentable; SCFA yield 7.9 mmol/g, but amylose films <50% fermentable; SCFA yield 1.4 mmol/g. Structural differences had a less apparent effect on the SCFA profile compared to composition. Less fermentable substrates, like wheat bran, pea hull and amylose films have the potential to produce a relatively high proportion of butyrate, albeit from a relatively low yield of SCFA. The ability to use resistant starch-based substrate structure to modify SCFA production during fermentation has implications for manipulating diet to help satisfy butyrate requirements for mucosal metabolism and associated anticarcinogenic properties throughout the colon. The problem to resolve is how substrates showing a low-level but sustained production of SCFA, and hence butyrate, can be manipulated to maintain threshold concentrations for mucosal health during transit in the colon.