Nanoemulsions present benefits such as an increase in the bioavailability, solubility and targeted delivery of encapsulated substances, and thus, they are a method of incorporating high nutritional value oils, such as high-oleic palm oil (HOPO). In this work, O/W nanoemulsions were obtained by microfluidization using HOPO (1–20% w/w) in the oily phase along with whey (1–20% w/w), Tween 20 (1:1 w/w ratio) and water in the aqueous phase following a surface response design methodology. The response variables were the average drop size (ADS), the polydispersity index (PDI), the zeta potential (ζ), CIELAB color parameters and viscosities of the fresh nanoemulsions (0 days) and nanoemulsions stored at two temperatures (5 and 19 °C) for 4 days. The ADS, PDI and ζ values varied between 163 and 2268 nm, 0.2 and 1, and − 29.7 and − 47.2 mV, respectively. The viscosity was affected by the storage temperature; after 4 days at 19 °C, it increased almost 6-fold compared to the viscosity of the fresh sample. With regards to the color parameters, significant changes were observed based on the concentrations of HOPO and whey. In addition, the prediction equations only presented errors below 7% for the evaluated variables, with R2 values above 0.85. Finally, the influence of whey protein denaturation at 60 °C on the stabilities of the two most stable nanoemulsions, according to the optimization process, was observed.