This study aimed to characterize serum lipidomic sexual dimorphism following acute exhaustive exercise under stringently controlled conditions. Forty healthy adults (20 males, 20 females) performed a CPET to exhaustion, with objective termination criteria and females tested during their mid-luteal phase. Untargeted lipidomics was performed on serum at baseline and at multiple postexercise time points. Males presented a greater absolute V ̇ O2peak (45.05 ± 4.14 vs 36.80 ± 3.69 ml/kg/min, P < 0.001) but a comparable V ̇ O2peak when FFM was normalized (56.50 ± 5.65 vs 55.45 ± 5.82 ml/kgFFM/min, P = 0.567). This standardized exhaustion was validated by both sexes achieving similar, high-intensity termination criteria (PRERpeak = 0.607, PRPE = 0.176, PHRpeak = 0.164). A total of 620 lipid species were identified via untargeted lipidomics. Sex-stratified analysis identified more significantly different lipids compared to pooled-sex analysis (male:179, female:288). Key sex discriminating lipids included PE 36:4, PC 32:1, SM d18:1/25:0, BMP 34:1, and PC 36:4. Cluster analysis revealed three recovery patterns, with lipid distribution differing significantly by sex. This study demonstrates that distinct metabolic response patterns exist between sexes when standardized exhaustion is achieved. Sex-stratified analysis identified more sex-specific lipids and pinpointed five sex-discriminating lipid molecular markers. The study revealed three different lipid recovery patterns, establishing that identical physiological endpoints arise from distinct metabolic mechanisms and underscoring the importance of sex-stratified analysis in exercise metabolism research.