Background: Alterations in hypothalamic–pituitary–adrenal (HPA)-axis activity, fatty acid metabolism, and their relation have been associated with (recurrent) major depressive disorder (MDD), although conflicting findings exist. Aims: To determine whether alterations in HPA-axis activity and fatty acids in recurrent MDD remain during remission (i.e. reflect a potential trait factor). Furthermore, to test the association between HPA-axis activity and fatty acids in patients versus controls. Methods: We cross-sectionally compared 73 remitted unmedicated recurrent MDD patients with 46 matched never-depressed controls. Measurements included salivary cortisol and dehydroepiandrosterone sulfate (DHEAS) (awakening, evening, and after sad mood induction) and erythrocyte fatty acid parameters: (I) three main fatty acids [omega-3 docosahexaenoic acid (DHA), and the omega-3 eicosapentaenoic acid/omega-6 arachidonic acid (EPA/AA)-ratio], and (II) structural fatty acid indices [chain length, unsaturation and peroxidation]. Results: Patients showed higher cortisol awakening responses (p = 0.006) and lower evening cortisol/DHEAS ratios (p = 0.044) compared to matched controls. Fatty acids did not differ between patients and controls, but HPA-axis indicators were significantly associated with fatty acid parameters in both groups (0.001 ≤ p ≤ 0.043). Patients and controls significantly differed in the relations between awakening DHEAS or cortisol/DHEAS ratios and fatty acid parameters, including unsaturation and peroxidation indices (0.001≤ p ≤ 0.034). Significance remained after correction for confounders. Conclusions: Our results further support alterations in HPA-axis activity, i.e. a lower baseline, but higher responsiveness of awakening cortisol, in remitted medication-free recurrent MDD patients. Furthermore, the relationship between HPA-axis and fatty acids showed significant differences in recurrent MDD patients versus controls. Prospective research is needed to determine the predictive value of this relationship for MDD recurrence.@en