Aim: Investigate the effect of restraint stress (RRS) and hind paw incisional injury, alone or in combination, on pain-, depression- and anxiety-related behaviour in female rats, and associated alterations in the endocannabinoid system. Methods: Thirty-two female Sprague-Dawley rats underwent either RRS (6h/day) or no stress (no-RRS) for 21 days prior to hind paw incision or sham surgery. Mechanical or heat hypersensitivity were assessed using Von Frey or Hargreaves’ tests, respectively. Anxiety- and depression-related behaviours were assessed using open field, elevated plus maze and sucrose splash tests. Spinal cord and brain regional levels of endocannabinoids and N-acylethanolamines were analysed post-mortem via LC-MS/MS. Data were analysed using either unpaired, two-tailed t-test or two-way ANOVA followed by Tukey’s post-hoc test. Results: No significant between-group differences were found in the anxiety- and depression-related tests. Increased mechanical and heat hypersensitivity were seen in the RRS+Incision group at post-surgical days 7-10 compared to noRRS+Incision. In the spinal cord, levels of anandamide (AEA) were lower in the RRS+Incision group compared to the noRRS+Incision group, and a between-subject effect of the interaction between stress and surgery was seen on levels of N-palmitoylethanolamide (PEA), N-oleoylethanolamide (OEA) and 2-Arachidonoylglycerol. Additionally, a between-subject effect of stress was seen on AEA and OEA levels in the PAG, and a stress x surgery interaction effect was seen on PEA levels in the PFC. Conclusions: RRS (6 h/day for 21 days) prior to incisional injury potentiates surgery-induced somatosensory hypersensitivity and results in alterations in spinal cord and brain regional endocannabinoid and N-acylethanolamine levels.