Oral ketone monoester supplementation does not accelerate recovery of muscle force or modulate circulating cytokine concentrations after muscle‐damaging eccentric exercise in healthy males and females

New Findings What is the central question of this study? Does acute ketone monoester supplementation enhance the recovery of muscle force and modulate circulating cytokine concentrations after muscle‐damaging eccentric exercise? What is the main finding and its importance? Ketone monoester supplementation increased plasma β‐hydroxybutyrate concentrations but did not attenuate the reduction in muscle force or the increase in plasma inflammatory cytokine concentrations that occurred after eccentric exercise. Notably we report novel data demonstrating a reduction in plasma TRAIL concentrations after eccentric exercise, highlighting TRAIL signalling as a possibly novel regulator of muscle recovery. Abstract Muscle‐damaging eccentric exercise is associated with inflammation and impaired muscle force. β‐Hydroxybutyrate (β‐OHB) reduces muscle protein breakdown during inflammation but whether oral ketone monoester supplementation accelerates recovery of muscle force after eccentric exercise is unknown. Sixteen healthy males and females consumed thrice daily ketone monoester (27 g per dose; n = 8; six females; KES) or isocaloric maltodextrin placebo (n = 8; four females; PLA) drinks (randomized, double‐blind, parallel group design) for 3 days beginning immediately after 300 unilateral eccentric quadriceps contractions during complete eucaloric dietary control (1.2 ± 0.1 g/kg BM/day standardized protein). Bilateral muscle force measurements and venous blood sampling were performed before and 3, 6, 24, 48 and 72 h after eccentric exercise. Plasma β‐OHB concentrations were greater in KES compared with PLA at 3 h (0.56 ± 0.13 vs. 0.22 ± 0.04 mM, respectively; P = 0.080) and 6 h (0.65 ± 0.41 vs. 0.23 ± 0.02 mM, respectively; P = 0.031) post‐eccentric exercise. Relative to the control leg, isokinetic work (by 20 ± 21% in PLA and 21 ± 19% in KES; P = 0.008) and isometric torque (by 23 ± 13% in PLA and 20 ± 18% in KES; P < 0.001) decreased from baseline at 3 h in the eccentrically exercised leg, and remained below baseline at 48 and 72 h, with no significant group differences. Of eight measured plasma cytokines, interleukin‐6 (P = 0.008) and monocyte chemoattractant protein‐1 (P = 0.024) concentrations increased after 6 h, whereas tumour necrosis factor‐related apoptosis‐inducing ligand concentrations decreased after 3 h (P = 0.022) and 6 h (P = 0.011) post‐exercise with no significant group differences. Oral ketone monoester supplementation elevates plasma β‐OHB concentrations but does not prevent the decline in muscle force or alter plasma inflammatory cytokine profiles induced by eccentric exercise.


INTRODUCTION
Unaccustomed eccentric exercise causes skeletal muscle damage, which is characterized by a rapid and protracted decline in muscle force and a parallel increase in muscle soreness and acute inflammation Pavis et al., 2021). Successful remodelling of damaged muscle and recovery of muscle force likely depend on the capacity to elevate post-exercise muscle protein synthesis rates to aid structural repair whilst limiting aberrant muscle protein breakdown in the face of acute inflammation Pavis et al., 2021). We recently reported that a dietary protein and polyphenol nutritional intervention approach that reduced acute intramuscular inflammation was associated with accelerated recovery of muscle force within 48 h following a single bout of eccentric exercise in healthy males and females . Indeed, there is a strong evidence base demonstrating that acute nutritional supplementation strategies can enhance the recovery of muscle force with 72 h of intense exercise (e.g., Cooke et al., 2009Cooke et al., , 2010Draganidis et al., 2017;Howatson et al., 2010;Michailidis et al., 2013), although granted this may not be beneficial for long term adaptation (Owens et al., 2019).
Elevations in circulating ketone bodies, via either increased endogenous production or exogenous intravenous infusion, have been associated with reduced muscle protein breakdown under various stress conditions including injury (Williamson et al., 1977), increased muscle protein synthesis signalling with protein ingestion following exercise (Vandoorne et al., 2017), energy restriction (Pawan & Semple, 1983) and inflammation (Thomsen et al., 2018). Indeed, intravenous administration of β-OHB achieving plasma concentrations of 3.5 mM decreases forearm phenylalanine rate of appearance (a measure of muscle protein breakdown) by 70% during acute endotoxaemia, resulting in a more positive net muscle protein balance in healthy males (Thomsen et al., 2018). Moreover, elevations in β-OHB are associated with a reduction in the activity of the NLRP3 inflammasome and downstream cytokine production in both cellular and animal models and in humans (Bae et al., 2016;Walsh et al., 2021;Youm et al., 2015).
Increasing plasma β-OHB nutritionally (in the absence of carbohydrate or energy restriction) may therefore be a novel and practical strategy to accelerate muscle recovery under inflammatory conditions.
We have previously demonstrated that circulating β-OHB concentrations can be increased to ∼3 mM in healthy, rested, non-energy restricted individuals via ingestion of the ketone monoester (R)-3-hydroxybutyl (R)-3-hydroxybutyrate, which is rapidly metabolized to the D-isoform of β-OHB (Myette-Côté et al., 2018;Neudorf et al., 2019). However, whether elevated plasma β-OHB via ketone ingestion can accelerate muscle force recovery and/or mitigate the systemic inflammatory response following muscle-damaging eccentric exercise remains unknown. The present study employed eccentric exercise to induce acute inflammation and a decline in muscle force to test the hypothesis that increasing circulating β-OHB via thrice daily oral ingestion of a ketone monoester drink could accelerate the recovery of muscle force in healthy, young volunteers undergoing conditions of full dietary control.

Ethical approval
Participants were informed of the experimental procedures, potential risks, and the purpose of the study before providing full written and informed consent. The study was approved by the Sport and Health Sciences Ethics Committee of the University of Exeter (Ref. No. 109703/A/01). The study conformed to the standard set by the Declaration of Helsinki, except for registration in a database.

Participants
Sixteen young, healthy males (n = 6) and females (n = 10) (age = 23 ± 3 years, BMI = 22 ± 3 kg/m 2 ) volunteered to take part in the present study. Participants attended the laboratory for a routine medical screening and completed a general medical questionnaire to assess their eligibility for participation. Exclusion criteria included a musculoskeletal injury, cigarette smoking, regular over-the-counter or prescribed medication use including non-steroidal anti-inflammatory medication, a diagnosed metabolic or cardiovascular impairment, routine use of nutritional supplements and a habitual protein intake of <0.8 g/kg BM/day assessed using a 3-day diet diary. Inclusion criteria included being aged 18-40 years, a BMI of >18 and <30 kg/m 2 and participating in recreational activity in the 6 months preceding the study (defined as exercise or sporting activities >2 h/week, but not structured exercise training such that participants were unaccustomed to maximal eccentric exercise). Female participants not using an oral contraceptive participated during days 6-12 of a regular menstrual cycle (i.e., mid-follicular phase).

Experimental protocol
Following screening and acceptance into the study, all participants attended a familiarization visit to familiarize them with dynamometry and muscle soreness measurement protocols. Familiarization to the eccentric exercise protocol consisted of just five submaximal repetitions so as not to induce any repeated-bout-effect adaptations.
At least 48 h following the familiarization visit, participants completed a 7-day experimental period under fully controlled dietary conditions (i.e., all food provided with set macronutrient profile based on individualized energy balance -details below and experimental protocol shown in Figure 1).

Eccentric knee extensor contractions
At ∼08.00 h on day 4 after a 10 h overnight fast, participants performed 300 (10 sets of 30 repetitions) voluntary maximal, unilateral, isokinetic, eccentric contractions of the knee extensors -a protocol we have previously demonstrated to impair muscle function and initiate inflammatory signalling in skeletal muscle within 24 h (Jameson, Kilroe, et al., 2021;Jameson, Pavis, et al., 2021;Pavis et al., 2021). Each contraction was performed at 60 • /s over an 80 • range of motion, which ended at full voluntary knee flexion. Each set was separated by 120 s of rest. Participants were instructed to resist the movement maximally throughout the full range of motion and verbal encouragement was provided throughout.

Muscle function testing
Muscle soreness and knee extensor peak isometric torque and total isokinetic work were determined as we have previously described Pavis et al., 2021) after a 10 h overnight fast at baseline and 24, 48 and 72 h after eccentric contractions.
Additional measurements of knee extensor peak isometric torque and total isokinetic work were performed 3 and 6 h after eccentric contractions at which time participants were 2 h postprandial. Lower body

New Findings
• What is the central question of this study?
Does acute ketone monoester supplementation enhance the recovery of muscle force and modulate circulating cytokine concentrations after muscledamaging eccentric exercise?
• What is the main finding and its importance?
Ketone monoester supplementation increased plasma β-hydroxybutyrate concentrations but did not attenuate the reduction in muscle force or the increase in plasma inflammatory cytokine concentrations that occurred after eccentric exercise. Notably we report novel data demonstrating a reduction in plasma TRAIL concentrations after eccentric exercise, highlighting TRAIL signalling as a possibly novel regulator of muscle recovery.

Dietary control
During the 7-day experimental period, participants received a fully controlled diet from the research team with energy requirements calculated as the basal metabolic rate (estimated via the Henry equations) (Henry, 2005) multiplied by an activity factor of 1.6 in order to maintain energy balance, as evidenced in our previous work Pavis et al., 2021Pavis et al., , 2022. Participants received all food products in individual packets and received stepby-step recipes. All meals and snacks were provided, whereas water and non-caloric drinks were allowed ad libitum. Caffeinated drinks were only permitted after completion of each testing visit. Daily protein intake was standardized at 1.2 g/kg BM/day (∼17% of energy intake), with the remaining calories being contributed by fat (∼33% of F I G U R E 1 Schematic representation of the experimental protocol. Participants were provided with full dietary control (1.2 g/kg BM/day dietary protein) for 6 days. Unilateral eccentric knee extensor contractions were performed at ∼08.00 h on day 4 (time = 0 h). Venous blood was sampled, and knee extensor isometric torque and isokinetic function were measured at baseline (day 3) and 3, 6, 24, 48 and 72 h after eccentric contractions. Muscle soreness was measured at baseline and 24, 48 and 72 h after eccentric contractions. Participants ingested either ketone monoester (n = 8) or energy-matched placebo (n = 8) drinks thrice daily on days 4, 5 and 6. energy intake) and carbohydrate (∼50% of energy intake). It has previously been demonstrated that administration of ketone monoester drinks with protein can potentiate muscle protein synthesis signalling pathways (Vandoorne et al., 2017). Thus, on day 4 participants consumed their controlled breakfast 1 h after eccentric exercise (providing 0.20 g/kg BM protein) and controlled lunch 1 h after muscle function testing at 3 h (providing 0.35 g/kg BM protein) to reduce the influence of nutrient distribution between individuals on acute recovery. Participants consumed their controlled diet ad libitum on days 1, 2, 3, 5 and 6. Compliance with the nutritional intervention was assessed via completed 6-day food diaries, returned food containers and daily communication with the participants. Non-compliance (i.e., consumption of food products not provided or failure to consume all products provided) was accounted for in the final dietary intake analysis.

Experimental drinks
Ketone and placebo drinks were taste-, texture-and energy-matched Participants were instructed not to consume food for 1 h following ketone monoester ingestion. Drinks were well tolerated, consumed within the allotted time (i.e., 5 min), and resulted in no reported adverse effects during or after the experimental period.

Blood sample collection and analysis
Ten millilitres of venous blood from the antecubital vein was collected at baseline and 3, 6, 24, 48 and 72 h after eccentric contractions.
Venous blood was collected into lithium heparin-containing tubes Rockville, MD, USA). All plates analysed for circulating cytokines had an average CV below 6% based on duplicate samples.

Statistics
In line with our previous work investigating the recovery of muscle

Participant characteristics and diet
Participants

Muscle soreness, total isokinetic work and peak isometric torque
Muscle soreness was not different between groups at baseline (P = 0.126) and increased from baseline in both groups after 24 h (from 8 ± 13 to 22 ± 15 mm in PLA and from 0 ± 0 to 17 ± 14 mm in KES; P = 0.000487), remained above baseline at 48 h (20 ± 6 mm in PLA and 18 ± 6 mm in KES; P = 0.0154) and was no longer different from baseline after 72 h in either group (P = 0.449; Figure 3) with the increase in muscle soreness not differing between groups (P = 0.747).
A single bout of 300 eccentric knee extensor contractions causes a transient decline in muscle force (Draganidis et al., 2016;Jameson, Pavis, et al., 2021;Michailidis et al., 2013;Paulsen et al., 2010;Pavis et al., 2021) which correlates with ultrastructural myofibrillar disruption . In the present study we used the established unilateral within-participant damaged versus control leg approach Pavis et al., 2021) and report relative to the contralateral control leg an immediate (3 h) decline in isometric torque and isokinetic work of ∼25% and ∼20%, respectively.
Characteristically of muscle damage, muscle force did not appreciably begin to recover until at least 48 h post-exercise (PLA group; Figure 4).  (Thomsen et al., 2018). The reason for a lack of effect of β-OHB on the recovery of muscle force in the present study is not clear, but it could be that our ketone dosing strategy was insufficient given that previous studies demonstrating an effect on muscle protein metabolism have used higher doses and achieved plasma β-OHB concentrations in excess of 3 mM (Thomsen et al., 2018;Vandoorne et al., 2017). Indeed, such doses affected leucine metabolism in particular (Vandoorne et al., 2017), which is thought to be essential for stimulating muscle protein synthesis, a prerequisite for recovery.
Thus, whilst we controlled for dietary protein and leucine intake, a limitation of the present study was that muscle protein synthesis or leucine signalling was not measured in the present study as we have done in the past Pavis et al., 2021). Moreover, the magnitude or context of inflammation caused by eccentric exercise versus endotoxaemia may be insufficient. Indeed the reduction in forearm muscle protein breakdown reported by Thomsen et al. during β-OHB and endotoxin infusion was accompanied by fever and markedly greater peak plasma IL-6 (3193 pg/ml) and IL-1β concentrations (Thomsen et al., 2018) whereas we observed peak IL-6 concentrations of just 1.6 pg/ml after eccentric exercise ( Figure 5b) and IL-1β was below the limit of detection, consistent with other studies that have failed to measure systemic changes in IL-1β after eccentric exercise (Cornish & Johnson, 2014;Hirose et al., 2004). Moreover, the ∼25% reduction in muscle force observed in the present study was less  than the ∼35-40% reduction in muscle force reported elsewhere following similar eccentric exercise protocols Paulsen et al., 2010;Pavis et al., 2021), perhaps because muscle function testing was performed 3 and 6 h after eccentric exercise, which we speculate would stimulate an increase in protein turnover that would help to promote muscle recovery Pavis et al., 2021). β-OHB did not suppress the increase in plasma IL-6 and MCP-1 measured 3-6 h post-exercise (Figure 5b, g) suggesting that β-OHB does not influence the acute systemic inflammatory response caused by eccentric muscle-damaging exercise.
We employed a rigorous dietary control and standardized protein intake to mitigate any effect differences in energy or macronutrient availability could have on plasma β-OHB within and between groups and the beneficial effect dietary protein intake can have on recovery from eccentric exercise (Buckley et al., 2010;Cockburn et al., 2008;Cooke et al., 2010;Draganidis et al., 2017;Jameson, Pavis, et al., 2021;Pavis et al., 2021). Furthermore, we chose to dose ketone monoester supplements thrice daily (as opposed to as a single post-exercise dose) to elevate plasma β-OHB for a sustained period of waking recovery and to complement the continual remodelling process that occurs for several days after eccentric exercise. There are, however, also some limitations to acknowledge. Firstly, we did not measure the time course of plasma β-OHB concentrations after ketone monoester supplementation. Therefore, the exact peak plasma β-OHB concentrations achieved in this participant cohort are not clear and can only be inferred based on our previous work in a different cohort supplementing with a similar ketone monoester dose (Myette-Côté et al., 2018). Moreover, whilst we analysed the plasma concentrations of a broad range of both pro-and anti-inflammatory cytokines, we did not investigate if ketone monoester supplementation modulated local inflammatory pathways that are known to be upregulated by eccentric exercise in skeletal muscle and mononuclear cells (García-López et al., 2007;Jameson, Pavis, et al., 2021). Indeed, we have shown that ketone monoester supplementation in individuals with obesity reduces lipopolysaccharide-stimulated monocyte caspase-1 activation without modulating systemic cytokine concentrations (Walsh et al., 2021). The insulinotropic effect of energy-matched placebo (25 g carbohydrate) and ketone monoester (26.7 g of ketone monoester) drinks was not measured and as such we cannot exclude an effect of insulin on post-eccentric exercise muscle protein turnover (Abdulla et al., 2016). We included both males and females in this study, with control for menstrual cycle phase, but the study was not powered to test for potential sex differences. Future mechanistic human studies in both males and females are required to ascertain the effect of ketone monoester supplementation on local inflammatory pathways.
In conclusion, oral ketone monoester supplementation in healthy males and females undergoing a strict dietary control resulted in sustained increase in plasma β-OHB but did not accelerate the recovery of muscle force in skeletal muscle damaged by eccentric exercise. Further, we report no impact of ketone monoester supplementation on plasma inflammatory cytokine profiles after eccentric exercise, but present novel data to demonstrate that a reduction in TRAIL signalling may be important in the remodelling of damaged skeletal muscle.

AUTHOR CONTRIBUTIONS
All experiments were performed in the Nutritional Physiology All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.