The purpose of this study was to evaluate the effects of microbial transglutaminase on the rheological and mechanical properties of gels from a commercial UHT milk made by acidification with D-gluconic-acid-δ-lactone. Although further heating of UHT milk, primarily carried out to inactivate the enzyme, showed a significant impact on gel firmness, we observed no enzyme-related effects at small deformation. The impact of the additional thermal treatment was also evident in large deformation measurements. In these experiments, gels from enzyme-treated milk revealed much higher elasticity and rupture force. Prolonged incubation time or increased enzyme concentration resulted in lower gel firmness (small deformation), indicating a reduced number of total bonds as a result of the restriction of proper rearrangements during acidification. Large deformation measurements revealed a heat-induced amplification of this effect, i.e., lower rupture forces at almost constant elasticity levels. In gels from milk, which was not subjected to thermal enzyme inactivation, we observed a further increase in both parameters. This is presumably caused by a combination of residual enzyme activity and of additional substrate, liberated during acidification.