Abstract In hypertrophic cardiomyopathy (HCM), cardiopulmonary exercise testing (CPET) is the gold standard for assessing exercise tolerance, with O2-pulse commonly used as a surrogate for stroke volume (SV). However, as SV reduction can be masked by increased oxygen extraction, direct non-invasive SV measurement is valuable. This study included 102 HCM patients (53±16 years, 78% male) with predominantly non-obstructive phenotype (74%) who underwent CPET with SV measurement using Physioflow® (PF). Abnormal O2-pulse kinetics were observed in 12 patients, all confirmed by abnormal SV trends with PF. Additionally, PF identified 28 more patients with altered SV kinetics. Abnormal SV trends were associated with higher peak VE/VO2 ratios (42.6 [37.4–47.5] vs. 38.0 [33.6–41.3]) and lower end-tidal CO2 values (31.8±4.9 mmHg vs. 34.3±5.6 mmHg, p<0.05). Patients with greater SV growth in the last 25% of exercise showed improved anaerobic threshold VO2 (49.8±12.3% vs. 43.9±15.2% predicted peak VO2), VO2/work slope (10.2±2.0 vs. 9.3±1.3 mL/min/Watt), and peak PetCO2 (34.5±5.6 mmHg vs. 32.3±5.2), alongside lower VE/VCO2 slope (28.7 [24.9–31.0] vs. 31.3 [27.3–34.2], p<0.05). Integrating PF and CPET enhances detection of abnormal SV kinetics, which are linked to reduced functional capacity in HCM patients.