Magnesium (Mg) plays a central role in neuronal activity, cardiac excitability, neuromuscular transmission, muscular contraction, vasomotor tone, and blood pressure, all of which are significantly related to physical performance. To date, the available data about detection of blood total Mg (tMg; free-ionized, protein-bound, and anion-complex forms) are inconsistent, and there is limited information on blood free-ionized Mg (Mg(2+)) in relation to physical exercise. The aim of this study was to determine the biochemical changes related to energy metabolism after acute exhaustive swimming exercise (AESE) in rats in an attempt to correlate the role of blood Mg(2+) with metabolites/enzymes related to energy production. After AESE, blood Mg(2+), tMg, K(+), partial pressure of carbon dioxide, lactate, total protein (T-PRO), high-density lipoprotein (HDL), creatinine (CRE), blood urea nitrogen (BUN), uric acid (UA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alanine phosphatase (ALP), lactate dehydrogenase (LDH), and creatinine kinase (CK) were significantly increased, whereas pH, partial pressure of oxygen, oxygen saturation, the Mg(2+)/tMg and Ca(2+)/Mg(2+) ratios, HCO3 (-), glucose, triglyceride (TG), and low-density lipoprotein (LDL) were significantly decreased. During AESE, lactate, T-PRO, albumin, AST, ALP, LDH, CK, CRE, BUN, and UA showed significant positive correlations with changes in blood Mg(2+), while glucose, TG, and LDL correlated to Mg(2+) in a negative manner. In conclusion, AESE induced increases in both blood Mg(2+) and tMg, accompanied by changes in blood metabolites and enzymes related to energy metabolism due to increased metabolic demands and mechanical damages.