Prolonged uninterrupted sitting of textgreater3-hours has been shown to acutely cause central and peripheral cardiovascular dysfunction. However, individuals rarely sit uninterrupted for textgreater2-hours, and the cardiovascular response to this time is currently unknown. In addition, whilst increased cardiorespiratory fitness (CRF) and habitual physical activity (HPA) are independently associated with improvements in central and peripheral cardiovascular function, it remains unclear whether they influence the response to uninterrupted sitting. This study sought to 1) determine whether 2-hours of uninterrupted sitting acutely impairs carotid-femoral pulse wave velocity (cfPWV), femoral ankle PWV (faPWV), and central and peripheral blood pressure, and (2) to investigate the associations between CRF and HPA versus PWV changes during uninterrupted sitting. Following 2-hours of uninterrupted sitting, faPWV significantly increased (mean difference [MD] = 0.26 m·s-1, standard error [SE] = 0.10, p = 0.013) as did diastolic blood pressure (MD = 2.83 mmHg, SE = 1.08, p = 0.014), however cfPWV did not significantly change. Whilst our study shows 2-hours uninterrupted sitting significantly impairs faPWV, neither CRF ( r = 0.105, p = 0.595) nor HPA ( r = -0.228, p = 0.253) were associated with the increases.