The purpose of this observational study was to examine the incidence of exercise-associated hyponatremia (EAH) in a 246-km continuous ultra-marathon. Over 2 years, 63 male finishers of the annual Spartathlon ultra-marathon foot race from Athens to Sparta, Greece were included in the data analysis. A blood sample was drawn from an antecubital vein the day before the race as well as within 15 min post-race and analyzed for sodium concentration. During the second year of data collection, blood was also drawn at the 93-km checkpoint ( = 29). Height and weight were measured pre and post-race. Mean race time of all subjects was 33 ± 3 h with a range of 23.5 and 36.0 h. Of the 63 finishers recruited, nine began the race with values indicative of mild hyponatremia. Seven runners were classified as hyponatremic at the 93-km checkpoint, three of whom had sodium levels of severe hyponatremia. After the race, 41 total finishers (65%) developed either mild ( = 27, 43%) or severe hyponatremia ( = 14, 22%). Mean change in bodyweight percentage and serum sodium from pre-race to post-race was -3.6 ± 2.7% (-2.5 ± 1.9 kg) and -6.6 ± 5.6 mmol·L, respectively. Pre-race serum sodium level was not a significant predictor of post-race serum sodium levels (β = 0.08, = 0.07, = 0.698), however, there was a significant negative association between change in bodyweight percentage and post-race serum sodium concentration (β = -0.79, = 0.29, = 0.011). The incidence of EAH of 52 and 65%, when excluding or including these individuals with pre-race hyponatremia, was the highest reported in current literature.