Background: White spot formation is one of the common side effects in orthodontic treatments and multiple enamel conditioning might happen even during on session of fixed orthodontic treatments. The aim of the present study was to evaluate the impact of multiple enamel conditioning with different methods on enamel micro‑, hardness (MH). Materials and Methods: In this In vitro experimental study, the buccal surfaces of 105 extracted premolars were evaluated in seven groups: One control and six experimental groups. The enamel conditioning was performed in three ways: Etching with phosphoric acid 37%, etching with phosphoric acid 37% followed by primer application and conditioning with self‑, etch primer. The conditioning process in each way was also performed twice consecutively. The specimens were submitted in pH cycling model with demineralization and re‑, mineralization solutions for 14 days. Afterward Vickers MH test was applied with 0. 981N force on the teeth for 10 s indentation time. Data were analyzed using One‑, Way ANOVA and Tukey HSD (honestly significant difference) test for multiple comparisons. A value of P < 0. 05 was considered statistically significant. Results: MH analysis showed statistically significant differences between the control group and the other conditioned groups (P < 0. 05). The groups conditioned with acid‑, etch and primer, particularly twice, showed the lowest amount of MH in comparison to other groups. Self‑, etch primer had the least effect on MH of the enamel. Single time etching without using primer, made no considerable difference when compared to multiple etching. Conclusion: Etching process and covering the enamel with primer decrease enamel MH. Using self‑, etch primer is a more conservative method of enamel conditioning.