Purpose <p>This study investigated thermoregulation and hydration dynamics in 80 runners (33 women, 41.3%; 47 men, 58.7%) during a 160-km ultra-endurance race with 9400 m of elevation gain in a tropical environment, where ambient temperatures ranged from 10.2 °C to 28.3 °C and relative humidity varied between 35 and 100%.</p> Methods <p>Core temperature (<i>T</i>_core; measured via ingestible telemetric capsules), body mass, fluid intake, and environmental conditions were recorded at key checkpoints. A linear mixed-effects model was used.</p> Results <p>Mean <i>T</i>_core was 37.9 ± 0.3°C (range 36.9–38.8 °C), with a peak of 38.9 ± 0.3 °C (range 38.1–39.96 °C). Faster runners exhibited higher <i>T</i>_core (<i>r</i> = –0.31, <i>p</i> = 0.024). While mean <i>T</i>_core was 37.9 ± 0.3 °C in male and 37.8 ± 0.3 °C in female, with peak values of 39.0 ± 0.4 °C and 38.7 ± 0.2 °C, respectively (<i>p</i> &lt; 0.05), sex was not a significant predictor in multivariate analysis. Mean body mass&#xa0;loss was −&#xa0;4.8%, with 31% of runners losing &gt; 6%, yet showed no correlation with <i>T</i>_core or performance. Key predictors of <i>T</i>_core were body mass index (BMI), age, speed, air&#xa0;temperature, humidity, radiation, race cumulative distance, and elevation changes (all <i>p</i> &lt; 0.05).</p> Conclusions <p>Ultra-endurance runners maintained core temperatures &lt; 40 °C despite significant body mass losses. These findings suggest that substantial body mass change may be a normal physiological adaptation during ultra-endurance running rather than a performance-limiting factor or a risk for hyperthermia. The study highlights the multifactorial nature of thermoregulation in ultra-endurance events and supports the need for individualized hydration strategies based on field data from prolonged, real-world conditions.</p> <p>The study was registered on ClinicalTrials.gov (NCT05098925).</p>