The elevated plus-maze (EPM) model usually employs nocturnal species (e.g. rats and mice) and the tests are almost exclusively performed during the diurnal phase (lights on), leading some laboratories to perform experiments with animals under a reversed light cycle to overcome this problem. However, it is questionable whether the artificial reversal of the light cycle for short periods guarantees modifications in all the physiological parameters found in normal subjects. The present study evaluated the session 1-session 2 (S1-S2) EPM profile in rats during their normal diurnal or nocturnal phase using different illumination conditions. Prior exposure to the EPM decreased open arm exploration for all groups in S2, regardless of the circadian phase and illumination condition; however, this behavior was decreased in subjects tested during the nocturnal phase, when compared to the diurnal phase. Risk assessment (RA) behavior was decreased under high illumination for both circadian phases in S1 and increased in the first minute of S2, when compared to the last minute of S1. Although open arm exploration and RA behavior were decreased under high illumination, when compared to low illumination conditions in both circadian phases, general locomotor activity was only decreased during the nocturnal phase. The results are discussed in terms of circadian variations in the behavioral profile and as a possible source of variability in pre-clinical models of anxiety.