(1) Background: Recent studies analyzed the participation and performance trends of historic races such as the oldest ultra-marathon (Comrades) or the oldest 100-km ultra-marathon (Biel). One of the toughest and historic ultra-marathons in the world is the ‘Spartathlon’ (246-km ultra-marathon from Athens to Sparta). The present study aimed to analyze the trends in participation and performance of this race. (2) Methods: Different general linear models were applied as follows: the first model was a two-way ANOVA (Decade × Sex), with separate models for all participants and for only the top five finishers in each race; the second model was a two-way ANOVA (Age Group × Sex); the third model was a two-way ANOVA (Nationality × Sex). (3) Results: Between 1982 and 2019, 3504 ultra-marathoners (3097 men and 407 women) officially finished the Spartathlon at least once. Athletes from Japan were the majority with 737 participants, followed by far by runners from Germany (n = 393), Greece (n = 326), and France (n = 274). The nations with the highest numbers of athletes amongst the top five performers were Japan (n = 71), followed by Germany (n = 59), and Great Britain (n = 31). Runners from the USA were the fastest in men, and runners from Great Britain were the fastest in women. Female and male runners improved performance across the decades. The annual five fastest women and men improved their performance over time. Runners achieved their best performance earlier in life (20–29 and 30–39 years) than female runners (30–39 and 40–49 years). Runners in age group 30–39 years were the fastest for all nationalities, except for Greece. (4) Conclusions: Successful finishers in the Spartathlon improved performance in the last four decades and male runners achieved their best performance ~10 years earlier in life than female runners.