The effect of pethidine as patient-controlled epidural analgesia (PCEA) on specific biochemical components in breast milk in relation to the timing of secretory activation is not well investigated. The aim of this study was to compare biochemical timing of secretory activation between women who had a vaginal (V) or Caesarean birth with pethidine-PCEA (CBP). Several milk samples were collected daily from 36 mothers (17 V, 19 CBP) for the first 265 h post-partum. Protein and lactose concentrations and Na+ and K+ ion levels were measured. Samples were assigned to three time periods: 0–72, >72–165 and >165–265 h post-partum for statistical analyses. Data were analyzed using linear mixed effect models. In the first 72 h post-partum, the mean difference in lactose concentration was 5 gL−1 higher in group V (P < 0.05). From >72–165 h post-partum, protein and Na+ concentrations were lower in group V (P = 0.05, P = 0.02), and K+ levels were higher in group V (P < 0.001). From >165–265 h post-partum, there were no significant differences between the groups. Biochemically, secretory activation had occurred by 72 h post-partum in both groups. There were greater variations in measured biochemical components observed within group CBP initially. However, by 165 h post-partum, there were no differences in the biochemical components between the groups. This suggests that effects of pethidine-PCEA are diminished by 72 h post-partum and undetected by 165 h.