Date of Submission



During prolonged (> 90 min), continuous steady-state exercise, skeletal muscle is fuelled by both carbohydrate (CHO) (i.e. muscle and liver glycogen, blood glucose and muscle, blood and liver lactate) and fat substrates (i.e. adipose and intramuscular triglycerides [IMTGs], blood-borne free fatty acids [FFAs] and TGs). The specific pattern of substrate oxidation is influenced by the relative exercise intensity, an individual’s training status and their preceding diet. However, it is well accepted that when exercising at high relative intensities (i.e. > 70% maximal oxygen uptake [V̇ O2max]), CHO-based fuels are the predominant fuel source. Despite CHO being important for sustaining prolonged exercise, recent attention has focused on exercise-nutrient protocols that reduce skeletal muscle dependence on CHO fuel sources and, instead, increase reliance on fat-based fuels. Such strategies include high-fat, low-CHO diets, training with low endogenous and exogenous CHO availability and oral ketone supplementation. In theory, strategies that “spare” the oxidation of CHO substrates should enhance endurance exercise performance. This thesis comprises a series of independent but related studies investigating the effects of manipulating both endogenous and exogenous fat availability on substrate metabolism, skeletal muscle adaptations and exercise performance.

Study 1 (described in chapter 4) investigated the effect of decreasing circulating FFA availability prior to and during half-marathon running. FFA availability was suppressed via the administration of nicotinic acid, ingested prior to and during exercise. The suppression of lipolysis and the exercise-induced rise in plasma FFAs did not impair half-marathon running capacity. When running at ~80% V̇ O2max for ~90 min there was a small but obligatory use of fat substrates, independent of CHO intake pre- and during exercise. However, CHO was the predominant fuel source, contributing between 80-90% to total energy expenditure.

Study 2 (described in chapter 5) examined the effects of ingesting a ketone diester on circulating ketone bodies, substrate metabolism and cycling performance under nutritional conditions replicating an elite professional cycling time-trial. Ketone ingestion increased circulating β-hydroxybutyrate and acetoacetate concentrations. Despite optimal nutritional support, the ketone diester was also associated with gut discomfort and an increased perception of effort, leading to an impairment of cycling time-trial performance.

Study 3 (described in chapter 6) manipulated endogenous fat and CHO availability via daily energy intake, to determine whether the metabolic perturbations from a high-fat diet are driven by high-fat or low-CHO availability. Participants consumed five days of a high-fat or highprotein diet (~65% energy intake), while ‘clamping’ CHO consumption to < 20% energy intake. When compared to an isoenergetic high-protein diet, five days’ adaptation to a high-fat diet resulted in greater whole-body rates of fat oxidation during submaximal cycling and decreased skeletal muscle mitochondrial respiration supported by octanoylcarnitine and pyruvate as well as uncoupled respiration at rest. Following one day of a high-CHO diet mitochondrial respiration returned to pre-diet, however whole body rates of substrate oxidation were only partially rescued.

This series of research studies contributes new knowledge to the literature by demonstrating that 1) fat substrates contribute < 20% to energy expenditure during prolonged, high-intensity running, independent of pre-exercise CHO intake 2) ketone diester ingestion impairs cycling time trial performance and is associated with a higher perception of effort, despite optimal nutritional feeding and 3) high dietary fat rather than low-CHO intake contributes to reductions in mitochondrial respiration and increases in whole-body rates of fat oxidation following a high-fat, low-CHO diet. However, this reduction can be partially rescued following one day of a high-CHO diet. This novel information provides evidence that high-fat diets and exogenous ketone drinks are not advantageous for an athletes training and competition due to their detrimental effects on substrate metabolism and skeletal muscle adaptations. Athletes should instead ensure high-CHO availability prior to and during competition to maximise whole-body rates of CHO oxidation rates.


Mary MacKillop Institute for Health Research

Document Type


Access Rights

Open Access


238 pages

Degree Name

Doctor of Philosophy (PhD)


Faculty of Health Sciences