Objective: The hypothalamic-pituitary-adrenal (HPA) axis evaluation has been conflicting in rheumatoid arthritis (RA) patients. Our aim was to evaluate the HPA axis response to the insulin tolerance test (ITT) in premenopausal female patients with RA before and after anti-tumor necrosis factor therapy (anti-TNF therapy). Design: A comparative cross-sectional analysis. Subjects and methods: Ten females with RA and without previous anti-TNF therapy were included. Five healthy females were included as controls. An ITT was performed before first dose of anti-TNF therapy and then after week 12. Anti-TNF therapy was applied every 14 days for 12 weeks. Cortisol and ACTH levels were measured at 0, 30, 45, and 65 min. Prolactin was measured at 0, 30, 45, 90, 120, and 150 min. Results: The ACTH basal plasma levels at weeks 0 and 12 did not show statistical differences, at 1.26 (0.41-2.12) vs 1.54 (0.60-2.49) respectively (PZ0.68). The controls demonstrated a higher ACTH response than in the RA patients at week 0 before the anti-TNF therapy (349.12 area under curve (AUC)), (PZ0.004) and a similar ACTH response to ITT to those of RA patients at week 12 after the use of the anti-TNF therapy (1087.42 AUC). Serum cortisol levels did not show significant changes when the ITT was performed before and after the anti-TNF therapy.