In this study, effect of temperature (4, 10, 25, and 35 degrees C) on microstructural and rheological properties of royal jelly (RJ) was investigated. Our results indicated that pH and proximate composition of RJ was insignificantly affected by temperature, but degrees Brix values increased. Microstructural images showed that RJ particles were homogeneous at 10 and 25 degrees C, but swelled at 35 degrees C. RJ samples showed shear thinning behavior with yield stress. The increasing temperature caused the increase of yield stress due to swelling of particles. All samples were characterized as weak gel-like behavior because G(') > G('') at up to 100 rad/s. Three interval thixotropic test (3ITT) showed that RJ samples could partially recover after high shear deformation, but increasing temperature decreased recovery percentage of samples. This study revealed that the temperature of RJ should never exceed to 10 degrees C to obtain better recovery during the processing. Practical applications Apicultural products such as royal jelly (RJ) have become very important nutritional products in recent times due to bioactive properties and are increasingly attracted by consumers. Bioactive properties of RJ can highly be deteriorated by process temperature and storage conditions. RJ has creamy and milk-like structure and contains high amounts of proteins and carbohydrates. Therefore, the rheological characterization of RJ can provide significant information about structure, freshness, end product quality for consumers, as well as process equipment design and energy consumption for manufacturers. The freshness of RJ is determined with the analysis of 10-HDA, furosine, superoxide dismutase and 57 kDa proteins. This study revealed that the rheological characterization of RJ at different temperatures may be helpful to estimate freshness and process conditions easier than analytical methods.