Motivation: The menopause is generally associated with lower cardiovascular function, which may affect cardiovascular adaptations to exercise training. Biomarkers to investigate such differences are often assessed under resting conditions (e.g. Nio et al., 2017), but this approach lacks insight into the human capacity to meet the cardiovascular demands of daily living. In addition, oestrogen has been suggested to upregulate slow L-type Ca2+ channels in the female basal subepicardium, but not in the basal subendocardium nor at the apex (Yang et al., 2012). This could manifest as differences in left ventricular (LV) rotational mechanics in vivo between pre- and post-menopausal women. The aim of this study was to compare the effects of exercise training on LV function and rotational mechanics between middle-aged pre- and post-menopausal women, specifically during acute cardiovascular challenges that mimic the activities of daily living. Methods: Twenty-five healthy untrained middle-aged women (age 45-58 years; 11 pre-menopausal, 14 post-menopausal) completed 12 weeks of high-intensity aerobic interval training (3 sessions/week consisting of 4 × 4-min intervals at 90-95% maximum heart rate). Before and after exercise training, (i) peak aerobic capacity and blood volume were determined, and (ii) LV function and rotational mechanics were assessed via echocardiography at -15 and -30 mmHg LBNP, and at 20, 40 and 60% peak exercise. Two-way ANOVAs were used to compare peak aerobic capacity and blood volume in pre- and post-menopausal women before and after exercise training, while three-way ANOVAs were used to examine LV function and rotational mechanics during physiological stress (α<0.1; covariate: age). The Benjamini and Hochberg method was used to control for false discovery rate (FDR). Results: Post-menopausal women had a smaller increase in peak aerobic capacity and blood volume after exercise training, compared with middle-aged pre-menopausal women (P<0.05; 9% difference in relative peak oxygen uptake aka VO2peak). Cardiac output, heart rate, systemic vascular resistance and LV volumes at rest and during physiological stress were similar between pre- and post-menopausal women (FDR-adjusted P>0.1). Peak basal rotation during exercise differed between pre- and post-menopausal women after exercise training (FDR-adjusted P=0.07; post hoc P=0.002; Figure 1). Conclusion: Post-menopausal women have a smaller increase in cardiorespiratory fitness after high-intensity aerobic interval training, compared with middle-aged pre-menopausal women. Acute cardiovascular challenges revealed differences between pre- and post-menopausal women at the LV base and not at the apex. Our findings provide new insight into the impact of the menopause on cardiac “functional” capacity.