The post‐operative period can generate immunological stress and can be modulated through supplementation with the omega‐3 series of polyunsaturated fatty acids. This study aimed to evaluate the effects of diets enriched with high doses of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids and glutamine on inflammatory mediators in dogs before and after ovariohysterectomy (OVH). Twelve female dogs were divided into two groups: group A was fed a commercial diet without the addition of EPA and DHA, and group B was fed an experimental diet enriched with EPA and DHA (0.2 g/100 kcal). Experimental diet intake initiated 21 days before surgery and continued until 30 days after OVH. Parameters measured were serum cytokines (TNF‐α, IL‐6 and IL‐10), C‐reactive protein (CRP), IGF‐1, lymphoproliferation and body composition before and after surgery. Statistical analyses were performed with SAS software considering the effects of age and diet and their interactions, and means were compared by the Tukey test. There was no difference between groups in body weight (p = .682), lean mass (p = .101) and body fat (p = .103). There were no group differences in serum concentrations of TNF‐α, IL‐6, IL‐10, IGF‐1, CRP and the percentage of lymphocyte proliferation. However, a time effect for TNF‐α was observed (p < .001), in which T0P (10 days after the surgical procedure) presented lower values of this cytokine when compared to the other evaluation time points; and interaction effects between group and time were observed for serum concentrations of IL‐6 (p < .001) and IL‐10 (p = .002). OVH procedure was not considered invasive enough to increase inflammatory cytokines after 30 days of surgery, as well as the dosage of the EPA and DHA used before and after the surgery did not modulate the inflammatory markers.