I-124-PET/CT can be used for pre-therapeutic assessment of radioactive iodine uptake in benign thyroid disorders, however systematic comparisons with intra-therapeutic uptake are still lacking for these disorders. The goals of this study were to compare I-124 RAIU and conventional I-131 RAIU tests with each other; to compare both tests with intra-therapeutic uptake (reference); and to verify the time course of radioactive iodine uptake at three time points (30, 102, and 336 h [14 days] post administration; p.a.). Thirteen patients with benign thyroid diseases underwent I-131 RAIU test and I-124 RAIU test one after another before the intra-therapeutic I-131 uptake (reference) was measured via short-range and long-range measurements. After correction for decay, relative uptake differences were calculated and subjected to the Bland-Altman method for the evaluation of levels of agreement. Radioactive iodine uptake tests with I-124-PET/CT and I-131 probe did not show systematic deviations at any time point. Likewise, at 30 and 102 h p.a. there was no systematic discrepancy between pre-therapeutic and intra-therapeutic uptake levels. At 14 days p.a., however, both pre-therapeutic tests tended to overestimate the uptake compared to reference. Findings showed, for the first time with I-124, that radioiodine therapy has some early radiobiological effects possibly limiting the accuracy of pre-therapeutic dosimetry. I-124 RAIU tests represent a feasible alternative to standard I-131 RAIU tests. The additional benefits of I-124-PET/CT (e.g., functional topography, inclusion of retrosternal areas, possibility to enable fusion imaging) may thus increase the scope of this technology in benign thyroid disorders.