Magnesium (Mg) plays an essential role as a regulator of membrane stability and is required for neuromuscular, cardiovascular, immune, and hormonal functions; it is also considered a potential limiting element for human performance. We examined the effects of long-term continuous endurance exercise on serum levels of Ca and Mg in professional cyclists during 21 days of competition. Fourteen male cyclists from two Pro Teams were recruited to participate in the study. The blood samples were collected at the following three points in time during the 3 weeks of La Vuelta a España: i) at rest, just before the beginning of the tour (baseline, T0), ii) after 9 days of racing (before the beginning of the 10th stage, T1) and iii) at the end of the tour (before the last stage, T2). Serum levels of Ca, Mg, creatinine, lactate dehydrogenase (LDH), creatinine kinase (CK), aspartate transaminase (AST), alanine transaminase (ALT), aldolase, and total proteins (TPs) were measured. The results of the study reveal a gradually decrease of Mg and Ca mean plasma levels through the 3 weeks of La Vuelta a España and an increase of the creatinine levels. Our findings suggest that duration, intensity, and training levels of exercise play decisive roles in the regulation of serum magnesium homeostasis after endurance exercise.