We evaluated the effects of a simulated workday of prolonged sitting on blood pressure (BP) and pulse wave velocity (PWV) and examined whether posture (seated vs. supine) affected responses. Participants ( n = 25) were adults, with overweight/obesity and elevated BP, and performed seated desk work for 7.5 h. BP and PWV were measured in seated and supine postures at baseline (7:15 a.m.), midday (12:05 p.m.), and afternoon (4:45 p.m.). Generalized linear mixed models evaluated the effects of prolonged sitting on BP and PWV within each posture and interactions by posture and sex. In the recommended postures, seated BP and supine carotid-femoral pulse wave velocity (cfPWV) and carotid-ankle pulse wave velocity (caPWV), but not carotid-radial pulse wave velocity (crPWV), significantly increased over the simulated seated workday (all p < 0.05; effect sizes [ d] ranged from 0.25 to 0.44). Whilst no posture-by-time interactions were observed ( p > 0.05), BP, caPWV, and crPWV were higher when seated versus supine (main effects of posture p < 0.05; d ranged from 0.30 to 1.04). Exploratory analysis revealed that females had greater seated BP responses ( p for interaction <0.05); seated PWV and supine BP and PWV responses were similar by sex ( p for interaction >0.05). A simulated workday of prolonged sitting increased seated BP and supine cfPWV and caPWV, and posture minimally influenced these responses. These results add to the evidence suggesting a deleterious effect of prolonged sitting on cardiovascular health.