ABSTRACT Purpose Despite a growing body of literature on the physiological responses to ultramarathon, there is a paucity of data in females. This study assessed the female physiological response to ultramarathon and compared the frequency of perturbations to a group of race- and time-matched males. Methods Data were collected from 53 contestants of an ultramarathon trail race at the Ultra-Trail du Mont-Blanc (UTMB®) in 2018/19. Before and within 2 h of the finish, participants underwent physiological assessments, including blood sampling for biomarkers (creatine kinase–MB isoenzyme [CK-MB], cardiac troponin I [cTnI], brain natriuretic peptide [BNP], and creatinine [Cr]), pulmonary function testing (spirometry, exhaled NO, diffusing capacities, and mouth pressures), and transthoracic ultrasound (lung comet tails, cardiac function). Data from eight female finishers (age = 36.6 ± 6.9 yr; finish time = 30:57 ± 11:36 h:min) were compared with a group of eight time-matched males (age = 40.3 ± 8.3 yr; finish time = 30:46 ± 10:32 h:min). Results Females exhibited significant pre- to postrace increases in BNP (25.8 ± 14.6 vs 140.9 ± 102.7 pg·mL −1 ; P = 0.007) and CK-MB (3.3 ± 2.4 vs 74.6 ± 49.6 IU·L −1 ; P = 0.005), whereas males exhibited significant pre- to postrace increases in BNP (26.6 ± 17.5 vs 96.4 ± 51.9 pg·mL −1 ; P = 0.002), CK-MB (7.2 ± 3.9 vs 108.8 ± 37.4 IU·L −1 ; P = 0.002), and Cr (1.06 ± 0.19 vs 1.23 ± 0.24 mg·dL −1 ; P = 0.028). Lung function declined in both groups, but males exhibited additional reductions in lung diffusing capacities (DL CO = 34.4 ± 5.7 vs 29.2 ± 6.9 mL⋅min −1 ⋅mm Hg −1 , P = 0.004; DL NO = 179.1 ± 26.2 vs 152.8 ± 33.4 mL⋅min −1 ⋅mm Hg −1 , P = 0.002) and pulmonary capillary blood volumes (77.4 ± 16.7 vs 57.3 ± 16.1 mL; P = 0.002). Males, but not females, exhibited evidence of mild postrace pulmonary edema. Pooled effect sizes for within-group pre- to postrace changes, for all variables, were generally larger in males versus females ( d = 0.86 vs 0.63). Conclusions Ultramarathon negatively affects a range of physiological functions but generally evokes more frequent perturbations, with larger effect sizes, in males compared to females with similar race performances.