Aim and Background: The wire’s stiffness might be changed over the usage time. The present in vivo study was conducted to investigate the effects of duration intervals on the stiffness of Nickel-Titanium (NiTi) orthodontic wires with different cross-sections to identify the appropriate size of wires. Materials and Method: The present clinical trial study was conducted on patients who were admitted to the Orthodontics Department of Tehran Dental Branch, Islamic Azad University. Totally, 90 samples of NiTi orthodontic wires, 30 samples with different sizes of 0.014, 0.016, and 0.018 inches were purchased. The measurement of the stiffness had been conducted before, after one month, and after two months of the treatment with 10 samples. The samples were taken to Razi Institute to evaluate archwires' stiffness, using a three-point bending test by Instron tester. Statistical analysis was conducted between and within-group differences using one-way ANOVA. Results: The stiffness of the 0.014, 0.016, and 0.018 inch-NiTi wires was insignificantly reduced from 64.0±0.8, 78.0±1.0, and 80.0±0.7 N/mm2 before being applied to the mouth to 63.0±3.0, 77.0±3.0, and 79.0±4.0 N/mm2 after one month and 62.0±2.0, 76.0±3.0, and 78.0±3.0 N/mm2 after two months of being used, respectively (P=0.8, P=0.8, and P=0.06, respectively). Conclusion: The stiffness of different cross-sections of NiTi wires did not show any significant changes during the 3 mentioned intervals.