The effects of heat treatment and incubation temperature on the rheological and microstructural properties of yogurt were studied. A central composite experimental design and response surface methodology were used for data analysis. The rheological properties were determined by dynamic low amplitude oscillation and the amount of spontaneous whey separation was quantified by the volumetric flask test. Confocal scanning laser microscopy was used to examine the gel structure. The storage moduli of yogurts increased with an increase in heating temperature and a decrease in incubation temperature. The maximum loss tangent value during gelation, permeability, and amount of spontaneous whey separation of yogurts increased with a decrease in heating temperature and with an increase in incubation temperature. These parameters indicated an increased possibility for rearrangements, which was confirmed by presence of large pores in the gel network. Second order polynomial models successfully predicted the effects of heating temperature and incubation temperature on the rheological properties, permeability, and whey separation of yogurts. Whey separation was negatively correlated with storage modulus (r= -0.66), and was positively correlated with the maximum loss tangent (r= 0.63) and permeability (r= 0.78). This study demonstrates that weak yogurt gels, which have high loss tangent values, favor rearrangements in the network and the resulting network has larger pores (high permeability) and exhibits greater whey separation.