Enrichment of food and beverages with bioactive lipids is an important initiative to improve consumer's health. Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids present in fish oil have been reported as those with the greatest bioactivity. Emulsions are an interesting alternative to incorporate functional oil; however, there are few studies on food microemulsions as a way to include this kind of compounds. The present work is intended to deepen the analysis of the Kolliphor RH40 emulsifier with potential application in food microemulsions, characterizing its micellar size and thermo-rheological properties, as well as analyzing the effect of environmental stress on physical and oxidative stability of a microemulsion containing fish oil. No significant changes in droplets size (<15 nm) or in their distribution was observed in a wide range of pH (3–9), ionic strength (0.1–10% CaCl2), centrifuging and different thermal treatments. During freezing, a slight increase in size (<21 nm) was detected, maintaining its optically transparent appearance. The high surface area of the microemulsion droplets led to the decrease in oxidative stability compared to fish oil in bulk. However, when microemulsions were stored at 4 °C, the EPA and DHA contents did not change during storage for 60 days. Display omitted •Emulsifiers derived from castor oil presented micelles size lower than 14 nm.•Thermo-rheological and flow behavior of emulsifiers was modeled.•Microemulsions were stable in a wide range of pH, ionic strength, and temperature.•Oxidation of microemulsions with fish oil increased with the storage temperature.•Refrigerated storage of microemulsions did not alter the content of EPA and DHA.