The aim of this study was to investigate the influence of isoenergetic substitution between the three energy delivering macronutrients in pre-starter diets on performance and intermediary nutrient metabolism in broiler chickens. From hatch until 5 days of age, 600 chicks, collected during peak of hatch, were fed one of the three experimental pre-starter diets with isoenergetic (13 MJ metabolisable energy/kg) substitutions between fat (43 vs. 108 g/kg), protein (126 vs. 240 g/kg) and carbohydrates (391 vs. 510 g/kg). After 5 days, commercial grower and finisher diets were provided. Pre-starter composition influenced body weight until slaughter age, although not statistically verifiable. Broilers fed the low protein (LP) pre-starter had the lowest body weight in relation to chickens on the low carbohydrate or low fat pre-starter diet. After hatch, chicks on the LP pre-starter diet were able to use the residual yolk sac more rapidly to fulfil their protein requirement, which is reflected in small intestine and liver development. Also, plasma metabolite levels were influenced mostly by the LP pre-starter, indicating that the main focus for the requirements of newly hatched chicks should be on proteins. Furthermore, optimal nutrition during the first day's post-hatch should take into account the contribution of the yolk.