ABSTRACT: A soy protein isolate (SPI) was thermally denatured at a critical concentration of 8% protein for 3 h at 95 °C, resulting in a powder that was readily reconstituted at ambient temperature and that demonstrated improved heat stability and cold-set gel functionality when compared to a control SPI. When SPI was heated at 3% protein equivalently, prior to reconstitution to 8% protein, the final viscosity was about 3 orders of magnitude less than the original sample. The viscosity of SPI heated at 3% protein was still nearly 2 orders of magnitude less than the original sample after both samples were reheated at 8% protein. These results suggested that heat denaturation at low protein concentrations limited network formation even after the protein concentration and interaction sites increased, impacting the isolate's cold gelling ability. Gelation was prevented upon treatment of SPI with iodoacetamide, which carbaminomethylated the cysteine residues, establishing the role of disulfide bonds in network formation. The viscosity of the 8% protein dispersion was also reduced by 2 orders of magnitude when treated with 8 M urea, and when combined with 10 mM DTT the gel viscosity was decreased by another order of magnitude. These results suggested that hydrophobic interactions played a primary role in gel strength after disulfide bonds form. The need for a higher concentration of protein during the heating step indicated that the critical disulfide bonds are intermolecular. Ultimately, the functionality produced by these protein–protein interactions produced a powdered soy protein isolate ingredient with consistent cold-set and thermal gelation properties.