Ozonated oils have demonstrated promising results for clinical applications. The reaction of ozone with the unsaturated compounds of oils produces by‐products such as ozonides and poly peroxides. A deeper knowledge of the dynamics of by‐product formation is helpful in determining the required ozonation degree to obtain therapeutic effects. The aim of this paper is to show the relationship between ozonation degree and structural and viscosity changes during the ozonation of grape seed (GS) and sunflower (SF) oils. Structural characterization was done by Fourier transform infrared (FT‐IR) and hydrogen‐1 nuclear magnetic resonance (1H NMR) spectroscopy, with iso‐ozonides being identified. Viscosity showed a significant increase during ozonation, a fact associated with poly peroxide formation. We have made use of the total unsaturation (TU) method to measure the ozonation degree. The TU of non‐ozonated GS oil was found to be higher than for SF oil (5.94 and 4.49 mmol per g of oil, respectively), and their by‐product distributions were also found to differ. In GS oil, three reaction steps were observed for double‐bond conversion into iso‐ozonides and poly peroxides, while the ozonides and poly peroxides were formed in parallel in SF oil. The studies we implemented characterized the differences in the reactivities of these oils with ozone. Practical applications: In this work, we propose using the TU method to measure the ozonation degree of ozonated oils. TU experimental determination is based on the ozonation of the sample, and the ozone‐oxidizable substrate is quantified. Despite GS and SF oils having similar fatty acid compositions, they contain other unsaturated compounds specific to their vegetal sources. These compounds are also reactive with ozone, and are also quantified by the TU method. The differences in distributions of by‐products among ozonated oils from different sources could explain why the ozonation degree need not be the same for different oils. Studies like this represent a feasibility foundation for controlling the therapeutic application of ozonated oils and correctly interpreting their well‐known clinical effects. Ozonated vegetable oils have many interesting applications in the food, cosmetic, and pharmaceutical industries, as well as in medicine. A deeper knowledge of the dynamics of by‐product formation is helpful in determining the required ozonation degree to obtain therapeutic effects. In this work, we propose using the total unsaturation method to measure the ozonation degree of ozonated oils. We showed the relationship between ozonation degree and structural and viscosity changes during the ozonation of grape seed and sunflower oils. The differences in distributions of by‐products among ozonated oils from different sources could explain why the ozonation degree need not be the same for different oils.

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