Ketone monoester ingestion increases postexercise serum erythropoietin concentrations in healthy men.

Intravenous ketone body infusion can increase erythropoietin (EPO) concentrations, but responses to ketone monoester ingestion postexercise are currently unknown. The purpose of this study was to assess the effect of ketone monoester ingestion on postexercise erythropoietin (EPO) concentrations. Nine healthy men completed two trials in a randomized, crossover design (1-wk washout). During trials, participants performed 1 h of cycling (initially alternating between 50% and 90% of maximal aerobic capacity for 2 min each interval, and then 50% and 80%, and 50% and 70% when the higher intensity was unsustainable). Participants ingested 0.8 g·kg-1 sucrose with 0.4 g·kg-1 protein immediately after exercise, and at 1, 2, and 3 h postexercise. During the control trial (CONTROL), no further nutrition was provided, whereas on the ketone monoester trial (KETONE), participants also ingested 0.29 g·kg-1 of the ketone monoester (R)-3-hydroxybutyl (R)-3-hydroxybutyrate immediately postexercise and at 1 and 2 h postexercise. Blood was sampled immediately postexercise, every 15 min in the first hour and hourly thereafter for 4 h. Serum EPO concentrations increased to a greater extent in KETONE than in CONTROL (time × condition interaction: P = 0.046). Peak serum EPO concentrations were higher with KETONE (means ± SD: 9.0 ± 2.3 IU·L-1) compared with CONTROL (7.5 ± 1.5 IU·L-1, P < 0.01). Serum ß-hydroxybutyrate concentrations were also higher, and glucose concentrations lower, with KETONE versus CONTROL (both P < 0.01). In conclusion, ketone monoester ingestion increases postexercise erythropoietin concentrations, revealing a new avenue for orally ingestible ketone monoesters to potentially alter hemoglobin mass.NEW & NOTEWORTHY To our knowledge, this study was the first to assess the effects of ketone monoester ingestion on erythropoietin concentrations after exercise. We demonstrated that ingestion of a ketone monoester postexercise increased serum erythropoietin concentrations and reduced serum glucose concentrations in healthy men. These data reveal the possibility for ketone monoesters to alter hemoglobin mass.

Nutritional regulation of hepatic de novo lipogenesis in humans.

PURPOSE OF REVIEW: De novo lipogenesis (DNL) is a metabolic process occurring mainly within the liver, in humans. Insulin is a primary signal for promoting DNL; thus, nutritional state is a key determinant for upregulation of the pathway. However, the effects of dietary macronutrient composition on hepatic DNL remain unclear. Nor is it clear if a nutrition-induced increase in DNL results in accumulation of intra-hepatic triglyceride (IHTG); a mechanism often proposed for pathological IHTG. Here, we review the latest evidence surrounding the nutritional regulation of hepatic DNL. RECENT FINDINGS: The role of carbohydrate intake on hepatic DNL regulation has been well studied, with only limited data on the effects of fats and proteins. Overall, increasing carbohydrate intake typically results in an upregulation of DNL, with fructose being more lipogenic than glucose. For fat, it appears that an increased intake of n-3 polyunsaturated fatty acids downregulates DNL, whilst, in contrast, an increased dietary protein intake may upregulate DNL. SUMMARY: Although DNL is upregulated with high-carbohydrate or mixed-macronutrient meal consumption, the effects of fat and protein remain unclear. Additionally, the effects of different phenotypes (including sex, age, ethnicity, and menopause status) in combination with different diets (enriched in different macronutrients) on hepatic DNL requires elucidation.

Intrahepatic triglyceride content: influence of metabolic and genetics drivers.

PURPOSE OF REVIEW: Intrahepatic triglyceride (IHTG) content is determined by substrate flux to, fatty acid synthesis and partitioning within, and triglyceride disposal from the liver. Dysregulation of these processes may cause IHTG accumulation, potentially leading to nonalcoholic fatty liver disease. The aetiology of IHTG accumulation has not been fully elucidated; however, environmental factors and heritability are important. Here, we review recent evidence regarding the contribution of metabolic and genetic components of IHTG accumulation. RECENT FINDINGS: Obesity and insulin resistance are the primary metabolic drivers for IHTG accumulation. These risk factors have pronounced and seemingly overlapping effects on all processes involved in determining IHTG content. The strong and interchangeable associations between obesity, insulin resistance and IHTG make it challenging to determine their relative contributions. Genome-wide association studies have identified a growing list of single nucleotide polymorphisms associated with IHTG content and recent work has begun to elucidate their mechanistic effects. The mechanisms underlying metabolic and genetic drivers of IHTG appear to be distinct. SUMMARY: Both metabolic and genetic factors influence IHTG content by apparently distinct mechanisms. Further work is needed to determine metabolic and genetic interaction effects, which may lead to more personalized and potentially efficacious therapeutic interventions. The development of a comprehensive polygenic risk score for IHTG content may help facilitate this.

Greater oxidation of dietary linoleate compared to palmitate in humans following an acute high-carbohydrate diet.

BACKGROUND: We have previously demonstrated that dietary saturated fatty acids (SFA), when compared to polyunsaturated fatty acids (PUFA), are preferentially partitioned into oxidation pathways. However, it remains unclear if this preferential handling is maintained when hepatocellular metabolism is shifted toward fatty acid (FA) esterification and away from oxidation, such as when hepatic de novo lipogenesis (DNL) is upregulated. AIM: To investigate whether an acute upregulation of hepatic DNL influences dietary FA partitioning into oxidation pathways. METHODS: 20 healthy volunteers (11 females) underwent a fasting baseline visit followed by two study days, 2-weeks apart. Prior to each study day, participants consumed an isocaloric high-carbohydrate diet (to upregulate hepatic DNL) for 3-days. On the two study days, participants consumed an identical standardised test meal that contained either [U13C]palmitate or [U13C]linoleate, in random order, to trace the fate of dietary FA. Blood and breath samples were collected over a 6h postprandial period and 13C enrichment in breath CO2 and plasma lipid fractions were measured using gas-chromatography-combustion-isotope ratio mass spectrometry. RESULTS: Compared to the baseline visit, fasting plasma triglyceride concentrations and markers of hepatic DNL, the lipogenic and stearyl-CoA desaturase indices, were significantly (p < 0.05) increased after consumption of the high-carbohydrate diet. Appearance of 13C in expired CO2 and tracer recovery were significantly (p < 0.05) higher after consumption of the meal containing [U13C]linoleate compared to [U13C]palmitate (5.1 ± 0.5% vs. 3.7 ± 0.4%), respectively. Incorporation of 13C into the plasma triglyceride and non-esterified fatty acid pool was significantly (p < 0.001) greater for [U13C]palmitate compared to [U13C]linoleate. CONCLUSION: Dietary PUFA compared to SFA appear to be preferentially partitioned into oxidation pathways during an acute upregulation of hepatic DNL, thus consumption of a PUFA-enriched diet may help mitigate intrahepatic triglyceride accumulation in individuals at risk of cardiometabolic disease.

Indirect Calorimetry-Based Novel Approach for Evaluating Metabolic Flexibility and Its Association with Circulating Metabolic Markers in Middle-Aged Subjects.

We propose a novel method for assessing metabolic flexibility (MF) through indirect calorimetry. A total of twenty healthy volunteers (10 females; 10 males) aged 45-65 were categorized into a Low-Intensity activity group (LI, 0-1 session of 1 h per week) and a High-Intensity activity group (HI, 5-6 sessions of 2 h per week). Volunteers underwent a stepwise exercise test on a cycle ergometer, connected to a calorimeter, to examine respiratory gas exchange to evaluate peak fatty acid Oxidation (PFO) and peak carbohydrate oxidation (PCO). Circulating peroxisome proliferator-activated receptor α (PPARα) biomarkers, docosahexaenoic acid/eicosapentaenoic acid (DHA/EPA) ratio and N-oleoylethanolamine (OEA), and the endocannabinoid- 2-arachidonoylglycerol (2-AG), were evaluated. We developed two MF parameters: the MF index (MFI), calculated by the product of PFO normalized per kg of fat-free mass (FFM) and the percentage of VO2max at PFO, and the peak energy substrates' oxidation (PESO), computed by summing the kilocalories from the PFO and PCO, normalized per kg FFM. The MFI and PESO were significantly different between the HI and LI groups, showing strong correlations with the circulating bioactive substances. Higher DHA/EPA ratio (p ≤ 0.05) and OEA (p ≤ 0.01), but lower 2-AG levels (p ≤ 0.01) were found in the HI group. These new parameters successfully established a functional link between MF and the balance of PPARα/endocannabinoid systems.

Dissociation between liver fat content and fasting metabolic markers of selective hepatic insulin resistance in humans.

OBJECTIVE: Fasting hyperglycemia and hypertriglyceridemia are characteristic of insulin resistance (IR) and rodent work has suggested this may be due to selective hepatic IR, defined by increased hepatic gluconeogenesis and de novo lipogenesis (DNL), but this has not been shown in humans. DESIGN: Cross-sectional study in men and women across a range of adiposity. METHODS: Medication-free participants (n = 177) were classified as normoinsulinemic (NI) or hyperinsulinemic (HI) and as having low (LF) or high (HF) liver fat content measured by magnetic resonance spectroscopy. Fractional gluconeogenesis (frGNG) and hepatic DNL were measured using stable isotope tracer methodology following an overnight fast. RESULTS: Although HI and HF groups had higher fasting plasma glucose and triglyceride concentrations when compared to NI and LF groups respectively, there was no difference in frGNG. However, HF participants tended to have lower frGNG than LF participants. HI participants had higher DNL compared to NI participants but there was no difference observed between liver fat groups. CONCLUSIONS: Taken together, we found no metabolic signature of selective hepatic IR in fasting humans. DNL may contribute to hypertriglyceridemia in individuals with HI but not those with HF. Glycogenolysis and systemic glucose clearance may have a larger contribution to fasting hyperglycemia than gluconeogenesis, especially in those with HF, and these pathways should be considered for therapeutic targeting.

Hepatic fatty acid and glucose handling in metabolic disease: Potential impact on cardiovascular disease risk.

The prevalence of metabolic diseases, including type 2 diabetes mellitus (T2DM) and metabolic dysfunction-associated steatotic liver disease (MASLD) is increasing. Although invariably associated with obesity, the importance of fat deposition in non-adipose tissue organs has yet to be fully explored. Pathological ectopic fat deposition within the liver (known as (MASLD)) has been suggested to underlie the development of T2DM and is now emerging as an independent risk factor for cardiovascular disease (CVD). The process of hepatic de novo lipogenesis (DNL), that is the synthesis of fatty acids from non-lipid precursors (e.g. glucose), has received much attention as it sits at the intersect of hepatic glucose and fatty acid handling. An upregulation of the DNL pathway has been suggested to be central in the development of metabolic diseases (including MASLD, insulin resistance, and T2DM). Here we review the evidence to determine if hepatic DNL may play a role in the development of MASLD and T2DM and therefore underlie an increased risk of CVD.

Breathlessness in a virtual world: An experimental paradigm testing how discrepancy between VR visual gradients and pedal resistance during stationary cycling affects breathlessness perception.

INTRODUCTION: The sensation of breathlessness is often attributed to perturbations in cardio-pulmonary physiology, leading to changes in afferent signals. New evidence suggests that these signals are interpreted in the light of prior "expectations". A misalignment between afferent signals and expectations may underly unexplained breathlessness. Using a novel immersive virtual reality (VR) exercise paradigm, we investigated whether manipulating an individual's expectation of effort (determined by a virtual hill gradient) may alter their perception of breathlessness, independent from actual effort (the physical effort of cycling). METHODS: Nineteen healthy volunteers completed a single experimental session where they exercised on a cycle ergometer while wearing a VR headset. We created an immersive virtual cycle ride where participants climbed up 100 m hills with virtual gradients of 4%, 6%, 8%, 10% and 12%. Each virtual hill gradient was completed twice: once with a 4% cycling ergometer resistance and once with a 6% resistance, allowing us to dissociate expected effort (virtual hill gradient) from actual effort (power). At the end of each hill, participants reported their perceived breathlessness. Linear mixed effects models were used to examine the independent contribution of actual effort and expected effort to ratings of breathlessness (0-10 scale). RESULTS: Expectation of effort (effect estimate ± std. error, 0.63 ± 0.11, P < 0.001) and actual effort (0.81 ± 0.21, P < 0.001) independently explained subjective ratings of breathlessness, with comparable contributions of 19% and 18%, respectively. Additionally, we found that effort expectation accounted for 6% of participants' power and was a significant, independent predictor (0.09 ± 0.03; P = 0.001). CONCLUSIONS: An individuals' expectation of effort is equally important for forming perceptions of breathlessness as the actual effort required to cycle. A new VR paradigm enables this to be experimentally studied and could be used to re-align breathlessness and enhance training programmes.

MLX plays a key role in lipid and glucose metabolism in humans: Evidence from in vitro and in vivo studies.

BACKGROUND AND AIM: Enhanced hepatic de novo lipogenesis (DNL) has been proposed as an underlying mechanism for the development of NAFLD and insulin resistance. Max-like protein factor X (MLX) acts as a heterodimer binding partner for glucose sensing transcription factors and inhibition of MLX or downstream targets has been shown to alleviate intrahepatic triglyceride (IHTG) accumulation in mice. However, its effect on insulin sensitivity remains unclear. As human data is lacking, the aim of the present work was to investigate the role of MLX in regulating lipid and glucose metabolism in primary human hepatocytes (PHH) and in healthy participants with and without MLX polymorphisms. METHODS: PHH were transfected with non-targeting or MLX siRNA to assess the effect of MLX knockdown on lipid and glucose metabolism, insulin signalling and the hepatocellular transcriptome. A targeted association analysis on imputed genotype data for MLX on healthy individuals was undertaken to assess associations between specific MLX SNPs (rs665268, rs632758 and rs1474040), plasma biochemistry, IHTG content, DNL and gluconeogenesis. RESULTS: MLX knockdown in PHH altered lipid metabolism (decreased DNL (p < 0.05), increased fatty acid oxidation and ketogenesis (p < 0.05), and reduced lipid accumulation (p < 0.001)). Additionally, MLX knockdown increased glycolysis, lactate secretion and glucose production (p < 0.001) and insulin-stimulated pAKT levels (p < 0.01) as assessed by transcriptomic, steady-state and dynamic measurements. Consistent with the in vitro data, individuals with the rs1474040-A and rs632758-C variants had lower fasting plasma insulin (p < 0.05 and p < 0.01, respectively) and TG (p < 0.05 and p < 0.01, respectively). Although there was no difference in IHTG or gluconeogenesis, individuals with rs632758 SNP had notably lower hepatic DNL (p < 0.01). CONCLUSION: We have demonstrated using human in vitro and in vivo models that MLX inhibition favored lipid catabolism over anabolism and increased glucose production, despite increased glycolysis and phosphorylation of Akt, suggesting a metabolic mechanism that involves futile cycling.

The effects of endogenously- and exogenously-induced hyperketonemia on exercise performance and adaptation.

Elevating blood ketones may enhance exercise capacity and modulate adaptations to exercise training; however, these effects may depend on whether hyperketonemia is induced endogenously through dietary carbohydrate restriction, or exogenously through ketone supplementation. To determine this, we compared the effects of endogenously- and exogenously-induced hyperketonemia on exercise capacity and adaptation. Trained endurance athletes undertook 6 days of laboratory based cycling ("race") whilst following either: a carbohydrate-rich control diet (n = 7; CHO); a carbohydrate-rich diet + ketone drink four-times daily (n = 7; Ex Ket); or a ketogenic diet (n = 7; End Ket). Exercise capacity was measured daily, and adaptations in exercise metabolism, exercise physiology and postprandial insulin sensitivity (via an oral glucose tolerance test) were measured before and after dietary interventions. Urinary ß-hydroxybutyrate increased by ⁓150-fold and ⁓650-fold versus CHO with Ex Ket and End Ket, respectively. Exercise capacity was increased versus pre-intervention by ~5% on race day 1 with CHO (p < 0.05), by 6%-8% on days 1, 4, and 6 (all p < 0.05) with Ex Ket and decreased by 48%-57% on all race days (all p > 0.05) with End Ket. There was an ⁓3-fold increase in fat oxidation from pre- to post-intervention (p < 0.05) with End Ket and increased perceived exercise exertion (p < 0.05). No changes in exercise substrate metabolism occurred with Ex Ket, but participants had blunted postprandial insulin sensitivity (p < 0.05). Dietary carbohydrate restriction and ketone supplementation both induce hyperketonemia; however, these are distinct physiological conditions with contrasting effects on exercise capacity and adaptation to exercise training.

The Effect of Blood Ketone Concentration and Exercise Intensity on Exogenous Ketone Oxidation Rates in Athletes.

INTRODUCTION: Exogenous ketones potentially provide an alternative, energetically advantageous fuel to power exercising skeletal muscle. However, there is limited evidence regarding their relative contribution to energy expenditure during exercise. Furthermore, the effect of blood ketone concentration and exercise intensity on exogenous ketone oxidation rates is unknown. METHODS: Six athletes completed cycling ergometer exercise on three occasions within a single-blind, random-order controlled, crossover design study. Exercise duration was 60 min, consisting of 20-min intervals at 25%, 50%, and 75% maximal power output (WMax). Participants consumed (i) bitter flavored water (control), (ii) a low-dose ß-hydroxybutyrate (ßHB) ketone monoester (KME; 252 mg·kg BW-1, "low ketosis"), or (iii) a high-dose ßHB KME (752 mg·kg BW-1, "high ketosis"). The KME contained a 13C isotope label, allowing for the determination of whole-body exogenous ßHB oxidation rates through sampled respiratory gases. RESULTS: Despite an approximate doubling of blood ßHB concentrations between low- and high-ketosis conditions (~2 mM vs ~4.4 mM), exogenous ßHB oxidation rates were similar at rest and throughout exercise. The contribution of exogenous ßHB oxidation to energy expenditure peaked during the 25% WMax exercise intensity but was relatively low (4.46% ± 2.71%). Delta efficiency during cycling exercise was significantly greater in the low-ketosis (25.9% ± 2.1%) versus control condition (24.1% ± 1.9%; P = 0.027). CONCLUSIONS: Regardless of exercise intensity, exogenous ßHB oxidation contributes minimally to energy expenditure and is not increased by elevating circulating concentrations greater than ~2 mM. Despite low exogenous ßHB oxidation rates, exercise efficiency was significantly improved when blood ßHB concentration was raised to ~2 mM.

ß-Hydroxybutyrate Oxidation in Exercise Is Impaired by Low-Carbohydrate and High-Fat Availability.

Purpose: In this study, we determined ketone oxidation rates in athletes under metabolic conditions of high and low carbohydrate (CHO) and fat availability. Methods: Six healthy male athletes completed 1 h of bicycle ergometer exercise at 75% maximal power (WMax) on three occasions. Prior to exercise, participants consumed 573 mg·kg bw-1 of a ketone ester (KE) containing a 13C label. To manipulate CHO availability, athletes undertook glycogen depleting exercise followed by isocaloric high-CHO or very-low-CHO diets. To manipulate fat availability, participants were given a continuous infusion of lipid during two visits. Using stable isotope methodology, ß-hydroxybutyrate (ßHB) oxidation rates were therefore investigated under the following metabolic conditions: (i) high CHO + normal fat (KE+CHO); (ii) high CHO + high fat KE+CHO+FAT); and (iii) low CHO + high fat (KE+FAT). Results: Pre-exercise intramuscular glycogen (IMGLY) was approximately halved in the KE+FAT vs. KE+CHO and KE+CHO+FAT conditions (both p < 0.05). Blood free fatty acids (FFA) and intramuscular long-chain acylcarnitines were significantly greater in the KE+FAT vs. other conditions and in the KE+CHO+FAT vs. KE+CHO conditions before exercise. Following ingestion of the 13C labeled KE, blood ßHB levels increased to ≈4.5 mM before exercise in all conditions. ßHB oxidation was modestly greater in the KE+CHO vs. KE+FAT conditions (mean diff. = 0.09 g·min-1, p = 0.03; d = 0.3), tended to be greater in the KE+CHO+FAT vs. KE+FAT conditions (mean diff. = 0.07 g·min-1; p = 0.1; d = 0.3) and were the same in the KE+CHO vs. KE+CHO+FAT conditions (p < 0.05; d < 0.1). A moderate positive correlation between pre-exercise IMGLY and ßHB oxidation rates during exercise was present (p = 0.04; r = 0.5). Post-exercise intramuscular ßHB abundance was markedly elevated in the KE+FAT vs. KE+CHO and KE+CHO+FAT conditions (both, p < 0.001; d = 2.3). Conclusion: ßHB oxidation rates during exercise are modestly impaired by low CHO availability, independent of circulating ßHB levels.

A Ketone Ester Drink Enhances Endurance Exercise Performance in Parkinson's Disease.

OBJECTIVES: Routine exercise is thought to be among the only disease-modifying treatments for Parkinson's disease; however, patients' progressive loss of physical ability limits its application. Therefore, we sought to investigate whether a ketone ester drink, which has previously been shown to enhance endurance exercise performance in elite athletes, could also improve performance in persons with Parkinson's disease. PARTICIPANTS: 14 patients, aged 40-80 years, with Hoehn and Yahr stage 1-2 Parkinson's disease. INTERVENTION: A randomized, placebo-controlled, crossover study in which each participant was administered a ketone ester drink or an isocaloric carbohydrate-based control drink on separate occasions prior to engaging in a steady state cycling test at 80 rpm to assess endurance exercise performance. OUTCOMES MEASURES: The primary outcome variable was length of time participants could sustain a therapeutic 80 rpm cadence. Secondary, metabolic outcomes measures included cardiorespiratory parameters as well as serum ß-hydroxybutyrate, glucose, and lactate. RESULTS: The ketone ester increased the time that participants were able to sustain an 80 rpm cycling cadence by 24 ± 9% (p = 0.027). Correspondingly, the ketone ester increased ß-hydroxybutyrate levels to >3 mmol/L and decreased respiratory exchange ratio, consistent with a shift away from carbohydrate-dependent metabolism. CONCLUSION: Ketone ester supplementation improved endurance exercise performance in persons with Parkinson's disease and may, therefore, be useful as an adjunctive therapy to enhance the effectiveness of exercise treatment for Parkinson's disease.

Nutritional Ketoacidosis During Incremental Exercise in Healthy Athletes.

Purpose: Ketosis, achieved through ingestion of ketone esters, may influence endurance exercise capacity by altering substrate metabolism. However, the effects of ketone consumption on acid-base status and subsequent metabolic and respiratory compensations are poorly described. Methods: Twelve athletically trained individuals completed an incremental bicycle ergometer exercise test to exhaustion following the consumption of either a ketone ester [(R)-3-hydroxybutyrate-(R)-1,3-butanediol] or a taste-matched control drink (bitter flavoured water) in a blinded, cross-over study. Respiratory gases and arterialised blood gas samples were taken at rest and at regular intervals during exercise. Results: Ketone ester consumption increased blood D-ß-hydroxybutyrate concentration from 0.2 to 3.7 mM/L (p < 0.01), causing significant falls versus control in blood pH to 7.37 and bicarbonate to 18.5 mM/L before exercise. To compensate for ketoacidosis, minute ventilation was modestly increased (p < 0.05) with non-linearity in the ventilatory response to exercise (ventilatory threshold) occurring at a 22 W lower workload (p < 0.05). Blood pH and bicarbonate concentrations were the same at maximal exercise intensities. There was no difference in exercise performance having consumed the ketone ester or control drink. Conclusion: Athletes compensated for the greater acid load caused by ketone ester ingestion by elevating minute ventilation and earlier hyperventilation during incremental exercise.

Beyond RPE: The Perception of Exercise Under Normal and Ketotic Conditions.

AIM: Subjective perceptions of exercising exertion are integral to maintaining homeostasis. Traditional methods have utilized scores of 'rating of perceived exertion' (RPE) to quantify these subjective perceptions, and here we aimed to test whether RPE may encompass identifiable localized perceptions from the lungs (breathlessness) and legs (leg discomfort), as well as their corresponding measures of anxiety. We utilized the intervention of ketoacidosis (via consumption of an exogenous ketone ester drink) to independently perturb exercise-related metabolites and humoral signals, thus allowing us to additionally identify the possible contributing physiological signals to each of these perceptions. METHODS: Twelve trained volunteers underwent two incremental bicycle ergometer tests to exhaustion, following ingestion of either an exogenous ketone ester or a taste-matched placebo drink. Cardiorespiratory measures, blood samples and perceived exertion scales were taken throughout. Firstly, two-way repeated-measures ANOVAs were employed to identify the overall effects of ketoacidosis, followed by generalized linear mixed model regression to isolate physiological predictors contributing to each perception. RESULTS: Rating of perceived exertion was found to contain contributions from localized perceptions of breathlessness and leg discomfort, with no measurable effect of ketoacidosis on overall exertion. Leg discomfort, anxiety of breathing and anxiety of leg discomfort were increased during ketoacidosis, and correspondingly contained pH within their prediction models. Anxiety of leg discomfort also encompassed additional humoral signals of blood glucose and ketone concentrations. CONCLUSION: These results indicate the presence of localized components of RPE in the form of breathlessness and leg discomfort. Furthermore, subjective perceptions of anxiety appear to result from a complex interplay of humoral signals, which may be evolutionarily important when monitoring exertion under times of metabolic stress, such as during starvation.