Nocturnal rodents should be assessed at an appropriate time of day, which leads to a challenge in identifying an adequate environmental light which allows animal visualisation without perturbing physiological homeostasis. Thus, we analysed the influence of high wavelength and low intensity light during dark period on physical exercise and biochemical and haematological parameters of nocturnal rats. We submitted 80 animals to an exhaustive exercise at individualised intensity under two different illuminations during dark period. Red light (> 600 nm; < 15lux) was applied constantly during dark period (EI; for experimental illumination groups) or only for handling and assessments (SI; for standard illumination groups). EI led to worse haematological and biochemical conditions, demonstrating that EI alone can influence physiological parameters and jeopardise result interpretation. SI promotes normal physiological conditions and greater aerobic tolerance than EI, showing the importance of a correct illumination pattern for all researchers that employ nocturnal rats for health/disease or sports performance experiments.Keywords: rats, chronobiology, aerobic tolerance, haematology, environmental illumination, exerciseMany authors have postulated the existence of circadian rhythms for physical exercise performance (12, 32), however, some of them do not entirely agree (10, 41). Despite lack of consensus, a common point raised over the years is the presence of confounding effects that disturb experimental control in human model investigations involving exercise chronobiology (12,17,32,41). In summary, confounding effects comprise social, nutritional, motivational and time-awake variations, masking the real chronobiological phenomena. In order to optimise such methodological control the animal model certainly becomes an interesting way. Rat circadian rhythms are well characterised by high core body temperature (25, 36), spontaneous activity (21), food intake, heart rate and locomotion (25) during dark period, representing 62% of total sleep time in the daylight period (38). Circadian modulations of these parameters are mainly synchronised by environmental light for these mammals (8,20,26). During the wakefulness period these nocturnal laboratory animals are usually kept under total darkness, promoting an optimum environment for the rat but adverse conditions for visualisation-based assessments. This condition encourages researchers to apply procedures under indiscriminate environmental light and time of day.