The purpose of this study was to evaluate the hydrocolloids–protein mixture as a fat replacer in sugar-free low-fat muffin cakes. In this study, a hydrocolloids mixture including konjac and guar gums and soy protein isolate (SPI) was applied to the cake. The combination of gums and SPI was named as mixture of stabilizers (MOS), and the treatments were designed using Design-Expert software and the response surface methodology (RSM) in order to optimize and reduce the oil content of muffin cakes by replacing it with MOS. Evaluation of treatments characteristics were investigated on days 1 and 15 of their production. The dependent variables were moisture content, water activity, specific volume, porosity, hardness, cohesiveness, springiness, chewiness and crumb color of cakes. The results show that increasing the percentage of MOS has positive effects on the final products in comparison to oil. In other words, an increase in the MOS content resulted in an increase in the moisture content, water activity, specific volume, height, springiness, cohesiveness, chewiness and L* (lightness) values, but negative effect on hardness, a* (redness) and b* (yellowness) values. As a result of optimizing using RSM, the usage of 4.08% oil and 0.31% MOS resulted in a 62.9% reduction in oil content in comparison with the control sample. The panelists assigned the lowest score to hardness and crumb color and the highest score to overall acceptability and cohesiveness to the optimized muffin.