Materials and Methods: An I-optimal response surface design was applied to investigate the effects of seed moisture content and microwave pretreatment time on oil content (%), PV, and OSI. Model adequacy was evaluated using analysis of variance and diagnostic statistical criteria. Optimal extraction conditions were identified through numerical optimization and experimentally validated. In addition, a 90-day storage stability study was conducted to compare oils produced under optimized and conventional conditions, monitoring PV and OSI changes during storage.

Results: The developed RSM models were statistically significant and showed strong predictive performance. Optimal conditions (approximately 8.05% seed moisture and 135.9 s microwave treatment) resulted in the highest oil yield while maintaining low PV and enhancing OSI. During storage, oil obtained under optimized conditions exhibited slower oxidative deterioration and greater resistance to lipid oxidation compared with conventionally extracted oil, indicating improved preservation of omega-3–rich polyunsaturated fatty acids, which are highly susceptible to oxidative degradation.

Conclusions: Microwave–moisture pretreatment optimization represents an effective strategy for producing high-quality chia seed oil with high content of omega-3 fatty acid, and improved oxidative stability during processing and storage, supporting its nutritional and health-related value for functional food and nutraceutical applications.