Sweat contained 260 compounds including 143 identified metabolites and technical replicates showed 14% median relative standard deviation. Identified compounds included amino acids, peptides, nucleotides, carbohydrates, xenobiotics, and cellular protectants.Previously identified compounds present in sweat included betaine, lactate, pyruvate, urea, caffeine,several amino acids, and others.Extended periods of exercise resulted in a 2-fold decrease in several metabolites associated with energy metabolism, such as lactate and pyruvate. Exercise reduced glucose levels ~30% at 40 and 60 minutes (p<0.05) in subjects drinking water while no significant decrease in glucose was observed when subjects drank the full calorie sports drink. Prolonged exercise caused a significant decrease (≥ 30%) of several amino acids reflecting the importance of acid-base balance and ammonia detoxification in muscle during exercise. A principal component analysis showed sweat composition from females was similar between the two sites. Prolonged exercise elicited a decrease in the sweat concentration of many metabolites at the end of exercise compared to sweat collected between 10-20min.