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It is well known that rats exhibit elevated levels of activity during the dark phase and reduced levels during the light phase of the photoperiod cycle. However, the information about the influence of the time-of-day on the strategies used to explore the environment is still not understood. Here we tested the hypothesis that time-of-day influences the fine-scale exploratory behaviour of rats, measured in the open field (OF) test, and emotionality of rats, measured in the elevated plus maze (EPM) test. Adult male Wistar rats were subjected to the OF and EPM tests during Morning, Afternoon, or Evening sessions. In the OF a principal component analysis (PCA) revealed that the Evening group exhibited longer duration of locomotion and rearing, and also higher distance travelled, trip length, inter-stop distance, number of stops and stops per trip compared to other groups. PCA also revealed that the Evening group exhibited shorter time spent at the home base, duration of locomotion along the perimeter and distance travelled along the perimeter compared to other groups. In the EPM test, there was no difference between the groups in any of the parameters evaluated. Our results indicate that the time-of-day may influence the spatio-temporal organization of exploration of rats subjected to unfamiliar environments. These alterations appear to be unrelated to differences in the emotional state of the animals. [ABSTRACT FROM AUTHOR]