Background: Little is known about the whole body oxidative stress burden following radioactive iodine (131I) therapy of thyroid diseases. Methods: We studied 17 patients with benign nodular goiter treated with 131I therapy. The targeted thyroid dose was 50 Gy in 11 patients pretreated with 0.1 mg of recombinant human TSH (rhTSH). In 6 patients, the applied thyroid dose was 100 Gy without rhTSH prestimulation. Well-established biomarkers of oxidative stress to RNA (8-oxo-7,8-dihydroguanosine; 8-oxoGuo) and DNA (8-oxo-7,8-dihydro-2'-deoxyguanosine; 8-oxodG) were measured in freshly voided morning urine (normalized against the creatinine concentration) at baseline, and 7 and 21 days after rhTSH (not followed by 131I), and 7 and 21 days after 131I therapy, respectively. Results: The baseline urinary excretions of 8-oxoGuo and 8-oxodG were 2.20 ± 0.84 and 1.63 ± 0.70 nmol/mmol creatinine, respectively. We found no significant changes in the excretion of any of the metabolites, neither after rhTSH stimulation alone nor after 131I therapy. Also, no significant differences were found between the rhTSH group (low dose, median 131I: 152 MBq) and the non-rhTSH group (high dose, median 131I: 419 MBq; 8-oxoGuo: p = 0.66, 8-oxodG: p = 0.71). Conclusion: Systemic oxidative stress, as detected by nucleic acids metabolites in the urine, is not increased after thyroid stimulation with 0.1 mg of rhTSH, or after 131I therapy. Our method cannot quantify the oxidative stress induced locally in the thyroid gland, but the study supports that 131I therapy of benign nodular goiter carries no or only a minute risk of developing subsequent malignancies. It remains to be explored whether our findings also apply to hyperthyroid disorders.