The health benefits of physical activity are recognized, however, high levels of exercise may lead to metabolic pathway imbalances that could evolve into pathological conditions like the increased risk of neurological disease observed in professional athletes. We analyzed the plasma/serum levels of 29 athletes from a professional soccer team playing in the Italian first league and tested the levels of psychophysical stress markers (vitamin D, creatine kinase, reactive oxygen species (ROS) and testosterone/cortisol ratio) during a period of 13 months. The testosterone/cortisol ratio was consistent with an appropriate training program. However, most of the athletes showed high levels of creatine kinase and ROS. Despite the large outdoor activity, vitamin D values were often below the sufficiency level and, during the “vitamin D winter”, comparable with those of the general population. Interestingly, high vitamin D values seemed to be associated to low levels of ROS. Based on the results of our study we proposed a vitamin D supplementation as a general practice for people who perform high levels of physical exercise. Beside the known effect on calcium and phosphate homeostasis, vitamin D supplementation should mitigate the high reactivity of ROS which might be correlated to higher risk of neurodegenerative diseases observed in professional athletes.