We aimed to investigate the effects of a single session of prolonged strenuous exercise (PSE) on arterial stiffness by measuring pulse wave velocity (PWV) before and after competition in an ultramarathon. A total of 20 routine ultramarathon competitors (UM) completed baseline and postrace evaluation of central PWV (cPWV), upper-limb PWV (uPWV), and lower-limb PWV (lPWV) using carotid artery - femoral artery, carotid artery - finger, and femoral artery - toe segments, respectively. Fourteen additional age- and gender-matched normally active participants (NA) took part in the identical baseline evaluation but did not participate in the race. Average ultramarathon completion time was 30 h 47 min. Mean arterial blood pressure was reduced after exercise (before exercise (pre), 92 ± 7 mm Hg; after exercise (post), 84 ± 7 mm Hg; P < 0.001), whereas heart rate was increased (pre, 57 ± 10 beats·min(-1); post, 73 ± 12 beats·min(-1); P < 0.001). Also, lPWV (pre, 11.8 ± 3.6 m·s(-1); post, 9.6 ± 2.6 m·s(-1); P < 0.05) and uPWV (pre, 5.0 ± 0.53 m·s(-1); post, 4.4 ± 0.8 m·s(-1); P < 0.01) were reduced after exercise. No change in cPWV occurred (pre, 4.1 ± 0.8 m·s(-1); post, 3.9 ± 1.3 m·s(-1); P = 0.55). At baseline, the NA group had significantly increased cPWV in comparison with the UM group (UM, 4.1 ± 0.8 m·s(-1); NA, 7.4 ± 1.3 m·s(-1); P < 0.001). Acute participation in PSE influenced peripheral but not central arterial stiffness. Those who routinely participate in PSE have reduced central arterial stiffness as compared with normally active, age- and gender-matched controls.