The monitoring of body mass (BM), plasma sodium concentration ([Na⁺]) and urinary specific gravity (Usg) are commonly used to help detect and prevent over- or dehydration in endurance athletes. We investigated pre-and post-race hydration status in 113 amateur 24-h ultra-runners, 100-km ultra-runners, multi-stage mountain bikers and 24-h mountain bikers, which drank ad libitum without any intervention and compared results of hyponatremic and non-hyponatremic finishers. On average, pre-race plasma [Na⁺] and both pre- and post-race levels of Usg and BM were not significantly different between both groups. However, nearly 86% of the post-race hyponatremic (exercise-associated hyponatremia, EAH) and 68% of the normonatremic (non-EAH) ultra-athletes probably drank prior the race greater volumes than their thirst dictated regarding to individual pre-race Usg levels. Fluid intake during the race was equal and was not related to plasma [Na⁺], Usg or BM changes. A significant decrease in post-race plasma [Na⁺], BM and an increasement in post-race Usg was observed in EAH and non-EAH finishers. Moreover, pre-race plasma [Na⁺] was inversely associated with post-race percentage change in plasma [Na⁺], and pre-race Usg and urinary [Na⁺] with percentage change in Usg in both groups with and without post-race EAH. Thirteen (11.5%) finishers developed post-race EAH (plasma [Na⁺] < 135 mM). The incidence of EAH in ultra-endurance athletes competing in the Czech Republic was higher than reported previously.