Acute exercise has been reported to increase thyroid hormone levels and decrease arterial stiffness in healthy young subjects. However, the effect of acute aerobic exercise on circulating thyroid hormone levels and arterial stiffness in patients with subclinical hypothyroidism remains unclear. The aim of this study was to investigate the effects of acute aerobic exercise on arterial stiffness and thyroid hormone levels, and any relationship between these endpoints, in patients with subclinical hypothyroidism. We studied patients with untreated subclinical hypothyroidism (n = 53, 65 ± 12 years old) compared with euthyroid subjects (n = 55, 64 ± 10 years old). Exercise analysis was performed with a ramp cycle ergometer test. Arterial stiffness (cardio-ankle vascular index, CAVI) was measured at baseline and 5 min after exercise. We collected participant blood samples for serum thyroid-stimulating hormone (TSH) and free thyroxine (FT4) measurements before and 5 min after exercise. The CAVI and serum TSH levels significantly decreased after exercise in the subclinical hypothyroidism group (CAVI; 8.1 ± 1.6 vs. 8.5 ± 1.5, p < 0.001, TSH; 6.7 ± 1.4 vs. 7.6 ± 1.2 μIU/ml, p < 0.001) and euthyroid group (CAVI; 7.6 ± 1.0 vs. 8.3 ± 0.9, p < 0.001, TSH; 2.2 ± 1.1 vs. 2.4 ± 1.2 μIU/ml, p = 0.005). The changes in CAVI from baseline compared with after exercise were lower, in absolute values, in the subclinical hypothyroidism group than in the euthyroid group (subclinical hypothyroidism group vs euthyroid group; ΔCAVI: − 0.4 ± 0.6 vs. − 0.7 ± 0.7, p = 0.012). The changes in serum TSH from baseline to after exercise were higher, in absolute values, in the subclinical hypothyroidism group than in the euthyroid group (subclinical hypothyroidism group vs euthyroid group; Δ serum TSH: − 1.3 ± 1.4 vs. − 0.3 ± 0.5, p < 0.001). The changes in CAVI from baseline to after exercise were negatively correlated with changes in TSH (r = − 0.32, p = 0.038) in the subclinical hypothyroidism group. In conclusion, acute aerobic exercise decreased both arterial stiffness and serum TSH levels in patients with subclinical hypothyroidism and euthyroid subjects. While the absolute change in arterial stiffness decreased, the absolute change in serum TSH levels increased in patients with subclinical hypothyroidism compared with euthyroid subjects. These data suggest that subclinical hypothyroidism reduces CAVI during acute aerobic exercise. Further changes in absolute levels of serum TSH in subclinical hypothyroidism may result in reduced CAVI improvement by acute aerobic exercise.