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To determine the incidence of and risk factors for exercise-associated hyponatremia (EAH) in cyclists completing a long-distance bike ride and to assess whether postexercise serum NT-proBNP concentration (brain natriuretic protein precursor) differed between riders with and without EAH.Observational study.“Around the Bay in a Day” cycle event, October 2010.One hundred thirty-nine cyclists prospectively enrolled, with 90 completing 210 or 250 km.Body weight change and fluid intake during the event, and postevent serum sodium concentration ([Na+]) and NT-proBNP concentration ([NT-proBNP]).Four riders (4.5%) were hyponatremic ([Na+] < 135 mmol/L). The lowest postride [Na+] was 126 mmol/L. Hyponatremia was associated with a mean weight gain of 3.4 kg (3.9% of total body weight). Significant negative correlations were found between postride [Na+] and change in weight (r = −0.34; P < 0.01) and fluid intake when expressed as total volume (r = −0.35; P < 0.01), mL/kg body weight (r = 0.33; P < 0.01), mL·kg−1·h−1 (r = −0.27; P < 0.01), or mL/h (r = −0.29; P < 0.01). NT-proBNP concentrations levels in 3 of the 4 hyponatremic subjects were markedly elevated compared with eunatremic subjects matched for age, sex, distance ridden, training, and medical history.Exercise-associated hyponatremia was found to occur in 4.5% of the study group and was associated with weight gain during a prolonged bike ride. Postride [Na+] varied inversely with weight change and with fluid intake. Three of 4 hyponatremic riders had significant elevations of [NT-proBNP]. These results support the hypothesis that overconsumption of hypotonic fluids in this setting is the most important cause of EAH.