Background Despite guidelines suggesting pancreatic enzyme replacement therapy (PERT) should be taken before or during a meal, it is currently unknown whether this has benefits over administration after a meal in individuals with cystic fibrosis (CF). Methods 18 children with pancreatic insufficient CF were randomised to two 13C-mixed triglyceride (13C-MTG) breath tests to assess lipase activity with PERT administered 10 min before and 10 min after a meal. Results were expressed as percentage cumulative dose recovered (PCDR) of 13CO2 and were compared with established values in healthy subjects. Gastric half emptying time (T½) was also assessed by a 13C-octanoate breath test. Results There was no difference in mean PCDR of 13CO2 between taking PERT before versus after the meal (p = 0.68). Eleven subjects had a greater PCDR when PERT was taken before and 7 when PERT was taken after the meal. 6/8 subjects (75%) with a lower than normal PCDR at one time point normalised PCDR when PERT timing was changed. When PERT was taken after the meal, PCDR was higher in normal vs. fast T½ (p = 0.04). Conclusions Changing PERT timing can result in normalised lipase activity. Gastric emptying rate may influence optimal timing of PERT. Clinical Trial Registration Number — This study was undertaken prior to the registration process being a commonly required practice.