Arterial stiffness, often measured by carotid-femoral pulse wave velocity (cfPWV), is a subclinical marker of cardiovascular disease that is known to be reduced by exercise training. Exercise is also known to have acute vascular effects, yet it is unclear whether exercise 24 h before cfPWV testing influences this outcome. Thirty healthy, young adults completed a supervised, 30-min bout of moderate-to-vigorous intensity treadmill running. cfPWV, systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR) were measured both before (after 48 h of abstaining from exercise) and 24 h after (with no additional exercise) the exercise session. From pre-exercise to 24 h post exercise, cfPWV decreased from 6.05±0.82 to 5.84±0.87 m s(-1) (P=0.02), SBP from 119.7±13.8 to 116.8±11.4 mm Hg (P=0.03) and DBP from 65.1±5.7 to 63.2±5.4 mm Hg (P=0.02), with no significant changes in HR. cfPWV was positively correlated with SBP pre-exercise (r=0.54, P<0.01) and post exercise (r=0.53, P<0.01). Changes in blood pressure explained 4-5% of the variability in cfPWV change; adjustments slightly attenuated the 24-h effects of exercise on cfPWV. Some evidence of gender differences was observed with higher cfPWV in males across assessments (P<0.05) and statistically significant reductions in cfPWV in males (-0.36±0.54 m s(-1) (P=0.02)) but not in females (-0.07±0.31 m s(-1) (P=0.41)). In conclusion, cfPWV decreased 24 h after an exercise bout, suggesting that exercise completed in the past 24 h should be considered before cfPWV testing.