PUFA modulate hypothalamic–pituitary–adrenal (HPA) axis activity and cortisol concentrations and therefore affect physiological stress responses and the regulation of energy balance in the short- and long-term. Especially dietary intake of n-3 PUFA and a lowered n-6:n-3 ratio are highly encouraged due to beneficial and diminishing effects on basal cortisol secretions. However, the time of such effects to occur and how plasma PUFA patterns affect cortisol concentrations in the short-term was rarely investigated. In order to address this, we supplemented forty male and forty female guinea pigs with diets high in the essential PUFA α-linolenic acid (ALA, 18 : 3n-3) and linoleic acid (LA, 18 : 2n-6) for 20 d. Saliva cortisol concentrations in relation to altering plasma PUFA patterns during this time span were analysed in a repeated measurement design both during basal conditions (individual housing) in 5-d intervals and during stressful social confrontations. We detected very fast plasma PUFA accumulation rates, corresponding to the major dietary PUFA, which resulted in plasma PUFA plateau phases after 10 d. ALA negatively and LA positively affected saliva cortisol concentrations throughout the study. A positive effect of the plasma n-6:n-3 ratio on saliva cortisol concentrations was detected during peak plasma PUFA accumulations and social confrontations, while no effects were detected in relation to plasma PUFA plateau phases. These results suggest that the plasma n-6:n-3 ratio diminishes HPA axis activity during altered physiological conditions only and highlights the importance of altering plasma PUFA patterns for HPA axis functions and the control of energy balance and physiological stress.