The main findings of this study were that melatonin has a significant ergogenic effect on the proposed exercise; however, it did not prevented either inflammation or tissue damage resulting from exhaustive exercise. Therefore, the initial hypothesis was only partially accepted, since a preventive effect of melatonin in exercised animals was not found, probably due to its massive ergogenic effect and features of the proposed exercise. As noted during the introduction section, it is well documented that intensity and duration of exercise influence a considerable number of physiological variables. Such a statement was enforced in our experiment, since the tlim leads to modulations in almost all of the studied parameters and seems to be confirmed with longer times to exhaustion (Tables 2 and 3).

In order to investigate the effect of exercise on local inflammation, we quantified proteins identified as the master controllers of inflammation24, IKK and IκB, responsible for the activity of the transcription factor κB (NFκB). The NF-κB is a key regulator of several biological systems and is associated with linking physiology to pathology when over-activated, its molecular pathway being considered the main inflammatory feedback mechanism in the body25. The activation of NF-κB depends on its translocation to the cell nucleus, where it acts in a pleiotropic fashion, influencing a large number of genes25. During the resting stage, NF-κB is sequestered in ambient cytosol by κB inhibitors (IκB)26, the main inflammatory protein detected in skeletal muscle of adult rats27. So, a high IκB content is interpreted as an anti-inflammatory parameter28. Stimuli such as high cytosolic calcium concentration29 and cytokines25,30 promote IκB kinase (pIKK) phosphorylation, which is responsible for disconnecting IκBα from NF-κB, eliciting its nuclear translocation and consequently increasing inflammation30. In this manner, disconnected IκBα molecules suffer ubiquitination and consequently a decreased level of this protein is found. IKK and IκB phosphorylation is increased by exercise in adult rat skeletal muscle, which invariably leads to local NF-κB activity27 and inflammatory feedback activation31.

In animals not submitted to exercise (experiment 1), melatonin did not modulate local or systemic inflammatory parameters (pIKKβ, IκBα, WBC and Lymp), but decreased skeletal muscle tissue damage marker (CK-MM, Fig. 2b), creatinine and urea (Table 1), exhibiting the protective features described in the literature7,8,11. However, the main contribution of our study is the interesting results regarding inflammatory and tissue damage markers in animals exercised until exhaustion during the light and dark periods after exposure to melatonin when compared to the control animals.

Despite the ergogenic effect of a single melatonin dose just before acute exercise remaining controversial in the literature8,32, our study found significantly enhanced performance in the animals that received this hormone (Fig. 2). As described, melatonin presents an anti-inflammatory function and anti-damage musculature effects7,8,11 and performance could be impaired by inflammation and tissue damage14,15,16. However, our results refute such statements in the context of exhaustive aerobic exercise since the systemic inflammation and tissue damage promoted in exercised animals in experiment 3 (all received melatonin) were apparently higher than in animals from experiment 2 (no melatonin). This apparent discrepancy in the inflammatory profile in melatonin treated animals could be associated with the different exercise protocols used in other studies. In addition, we observed that the CK-MM increased by 151.6% in the NEx group when compared to the NCt group, but this outcome in the NMEx group was 324.05% higher than in the NM group. Neutrophil counts, an acute exercise-induced inflammation marker, increased 90.68% in the NEx vs NCt group, but were 180.29% higher in the NMEx group compared to NM. Because the NMEx rats swam 126 minutes (155.84%) longer than the NEx group at the same intensity due to melatonin´s massive ergogenic effect, it is clear why higher inflammation and tissue damage were found in animals that swam longer. Therefore, we introduced this paradoxical melatonin effect on adults swimming rats exercised until exhaustion at anaerobic threshold intensity. Melatonin enhanced the performance but also the inflammation and damage to skeletal muscle tissue. Despite partially denying our initial hypothesis, high levels of inflammation and tissue damage are also found in marathon and ultramarathon athletes and the relationship between performance and tissue damage and inflammation remains under investigation33,34.

In general, interpreting all of the physiological, metabolic, oxidative, inflammatory and tissue damage markers assessed in our study, the exercise duration was increased by the effect of melatonin and led to more alterations. Our results suggested that the ergogenic effect of melatonin is significantly stronger than its protective effect with respect to exercise at anaerobic threshold performed until exhaustion, taking the exercise duration as responsible for masking the protective effect of melatonin. Future studies should reproduced this experiment design but employing limits on the exercise duration in order to elicits investigation of the protective effect of melatonin, better understand the mechanistic of melatonin´s ergogenic effect and its role as an anti-inflammatory, antioxidant and tissue damage preventing agent in long duration aerobic exercises.