Combined isometric exercise or metaboreflex activation (post-exercise muscle ischaemia (PEMI)) and cold pressor test (CPT) increase cardiac afterload, which may lead to adverse cardiovascular events. l-Citrulline supplementation (l-CIT) reduces systemic arterial stiffness (brachial-ankle pulse wave velocity (baPWV)) at rest and aortic haemodynamic responses to CPT. The aim of this study was to determine the effect of l-CIT on aortic haemodynamic and baPWV responses to PEMI+CPT. In all, sixteen healthy, overweight/obese males (age 24 (sem 6) years; BMI 29·3 (sem 4·0) kg/m2) were randomly assigned to placebo or l-CIT (6 g/d) for 14 d in a cross-over design. Brachial and aortic systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP), aortic augmented pressure (AP), augmentation index (AIx), baPWV, reflection timing (Tr) and heart rate (HR) were evaluated at rest and during isometric handgrip exercise (IHG), PEMI and PEMI+CPT at baseline and after 14 d. No significant effects were evident after l-CIT at rest. l-CIT attenuated the increases in aortic SBP and wave reflection (AP and AIx) during IHG, aortic DBP, MAP and AIx during PEMI, and aortic SBP, DBP, MAP, AP, AIx and baPWV during PEMI+CPT compared with placebo. HR and Tr were unaffected by l-CIT in all conditions. Our findings demonstrate that l-CIT attenuates aortic blood pressure and wave reflection responses to exercise-related metabolites. Moreover, l-CIT attenuates the exaggerated arterial stiffness response to combined metaboreflex activation and cold exposure, suggesting a protective effect against increased cardiac afterload during physical stress.