*3.2. High-Fat Diets*

High-fat diets (HFD) have been widely applied for decades as a treatment option for certain diseases such as epilepsy or as an effective dietary strategy for weight loss [124]. In recent years, these diets have also become widespread in endurance athletes [14–19,21–24,38]. High-fat diets applied in the athletic population are grouped under two main categories: (1) a ketogenic low-CHO high-fat (K-LCHF) diet, and (2) non-ketogenic high-fat (NK-LCHF) diet (described in Table 2). While a ketogenic diet aims to increase blood ketone levels from 0.5 to 3.0 mmol/L, non-ketogenic diets aim to provide potential benefits without reaching higher blood ketone concentrations. Ketosis is considered as a survival mechanism for the body to equilibrate blood glucose during a metabolic crisis, such as a lack of calories or glucose, in fasting conditions, or prolonged exercise and to provide energy to the brain, whose survival depends on ketone body (KB) utilization in case of glucose deprivation [125].


**Table 2.** Types and application processes of new diets applied by endurance athletes.


CHO: carbohydrate, FODMAP: fermentable oligosaccharides, disaccharides, monosaccharides and polyols.

> In HFD studies on endurance athletes, K-LCHF diets have been commonly applied for diet periods ranging from three to 12 weeks [16–19,21–23,38]. Two studies, a case report (a 10 week K-LCHF diet) [21] and a cross-sectional study (a 20-month K-LCHF diet) [24], examined the effects of longer-term ketogenic diets on performance. For NK-LCHF diets, three studies, two crossover (a 2 week NK-LCHF diet) [26,28] and a cross-sectional (6-mth NK-LCHF diet) study [27], also investigated the impact of NK-LCHF diets on performance and lipoprotein profiles in endurance athletes (Detailed in Table 1). Besides these ketogenic diet applications, acute [30–39] or long-term [130] administration of KBs (in a ketone ester (KE) or ketone salt (KS) form) and CHO restoration following keto-adaptation [26,40–43,45–47] have also been evaluated in endurance athletes. Additionally, studies have been conducted to investigate the effects of an acute pre-exercise high-fat meal [51], and a short-term (1.5 days) fat supplementation during high-CHO diet

administration [49,50]. In this section, we will discuss these high-fat studies in detail, with all their beneficial and harmful consequences for endurance athletes.

#### 3.2.1. Potential Beneficial Aspects of High-Fat Diets

High-fat diet administration has taken place in endurance athletes with the aim of improving the utilization of fatty acids and KB [14,19,20,24–26,28,32–36,41–43,45–47,49–51], sparing muscle glycogen stores [24,37,42,44,46,47], increasing weight loss, especially body fat mass [14,19,21,28], improving aerobic capacity [28], improving time to exhaustion [26,51] and time-trial performance [33,46,131], regulating performance-related parameters [34,36,39], increasing cognitive performance [38], regulating exercise-associated immunologic and hormonal response [15,22,30], increasing cellular gene expression [132], and attenuating overreaching syndrome [130].

One of the main goals of applying a high-fat diet to improve performance is to increase the body's ability to use KB and fatty acids as an energy source [14,19,20,24–26,28,32–36,41– 43,45–47,49–51]. The enhancement of the body's ability to use KB as an energy source generally occurs in two type manipulations: (1) By restricting dietary CHO intake for a prolonged time, the body adapts metabolically to using KB instead of glucose; this process is called keto-adaptation [24]. (2) Acute KB supplementation instantly changes fuel usage from CHO to KB [30–39,130].

Improvement of fat utilization to fuel, especially during prolonged exercise, may provide advantages for endurance athletes, including the glucose-sparing effect that, in particular, has vital importance for the brain during times of glucose depletion [133]. While the intramuscular triglyceride stores are predominantly preferred to provide energy during low- to moderate-intensity exercise (50–75% VO2max), in moderate to vigorous-intensity exercises (>75% VO2max), muscle glycogen is used as the primary substrate to obtain energy provisions [134]. However, since the substrate utilization highly depends on the diet pattern, keto-adaptation results in a shift from glycogen to FFA or KBs, even during high-intensity exercises [21]. A number of studies such as K-LCHF [14,15,19–21,24,25] and NK-LCHF trials [26,28], acute KB administration [32–36,39], keto-adaptation followed by CHO loading [41–43,46], and pre-workout HF meal administration [51] proved that fat oxidation significantly increased at rest and during exercise after HFD applications. Only studies practicing the short-term fat administration during high-CHO diet administration in trained male cyclists revealed that overall fat oxidation did not alter during prolonged exercise and during submaximal or one hour time-trial (TT) exercise training [49,50]. However, one of the studies noted that fat oxidation significantly increased regardless of diet [50], while another highlighted that intramyocellular lipid utilization increased 3-fold in the fat supplemented group [49]. Taking all studies together, it seems that all applications aiming to increase fat ingestion provide better fat and KB utilization in the body, especially during exercise. This metabolic advantage appears to be unique for enhancing endurance performance.

However, along with the changes in substrate utilization towards fatty acids and KBs, KD might not be advantageous for exercise that highly relies on anaerobic metabolism and requires glucose flux such as short-duration exercise or long-duration exercise with interval sprints. In a randomized, crossover study in trained endurance athletes, it was stated that a 5 day fat adaptation followed by 1 day CHO restoration caused a decrease in glycogenolysis and PDH activation [47]. The findings suggested that this dietary manipulation could result in an increase in the NADH/NAD+ ratio or the Acetyl-CoA/CoA ratio, which could result in sustained attenuation of PDH activity and impaired glycolysis metabolism. Further research should be elucidated on the possible interaction between impaired glycolysis metabolism and ketogenic diets on prolonged exercise with anaerobic metabolism or high-intensity intermittent exercise.

As it is well known that depleting glycogen stores is one of the major causes of fatigue during endurance exercise [2], HFD also aims to reduce muscle glycogen utilization to ensure CHO availability for longer periods of time during endurance training. Although one study on endurance-trained male cyclists showed that muscle glycogen utilization significantly decreased after a 10 day fat adaptation followed by 3 day CHO restoration trial compared to a high-CHO trial [46], others investigating muscle glycogen utilization claimed that no difference was observed between the intervention and the control trial [24,37,42,47]. In addition, a cross-sectional study on male endurance runners stated that muscle glycogen utilization did not alter after an average of a 20-month K-LCHF or high-carbohydrate (high-CHO) diet. Therefore, studies on HFD and its "muscle glycogen sparing effect" remain controversial. We cannot conclude that HFD provides an advantage to spare muscle glycogen during endurance training. Further work is needed to assess muscle glycogen utilization.

K-LCHF diets might be an effective option for athletes who aim to lose body weight (BW) and body fat while sparing muscle mass [14,19,21,28]. A crossover study assessing the effects of a long-term (4 week) K-LCHF diet rich in polyunsaturated fatty acids on aerobic performance and exercise metabolism in trained off-road cyclists revealed that BW and body fat percentage decreased after long-term KD [28]. It was also stated that the long-term K-LCHF diet improved maximum oxygen consumption and decreased post-exercise muscle damage. The findings sugges<sup>t</sup> that a long-term K-LCHF diet may provide advantages to both body composition and endurance performance. However, another study claimed that long-term KD (for 12 weeks) caused a decrease in both body fat percentage (5.2%) and body mass (5.9 kg) in endurance-trained athletes [14]. However, results also showed that although long-term KD resulted in improved body composition, it had no impact on 100 km TT performance. Consistent with this study, Heatherly et al. [19] investigated the impact of a 3 week ad libitum ketogenic diet on markers of endurance performance in recreationally competitive male runners. Results showed that the body composition of subjects positively changed with a decrease of ~2.5 kg BW and skinfold thickness occurring at multiple sites in the trunk region. However, KD did not affect exercise-induced cardiorespiratory, thermoregulatory, and perceptional responses and 5 km TT performance, and perceived exertion [19]. Findings indicate that KD may be an alternative strategy for reducing fat mass regardless of endurance performance.

On the other hand, Zinn et al. [21] investigated the 10 week ketogenic diet experiences of five endurance athletes and the effects of this diet on body composition and exercise performance. Although body mass and the sum of skinfolds were reduced by an average of 4 kg and 25.9 mm, respectively, endurance athletes experienced an inability to maintain high-intensity exercises during this period [21]. These findings raised doubts about the use of KD for weight loss in endurance athletes. In addition to that, a recent study compared the efficiency of two energy-reduced ( −500 kcal·day−1) diets, including a cyclical ketogenic reduction diet (CKD), defined as a high-fat low-CHO (>30 <sup>g</sup>·day−1) diet for five days, followed by a high carb diet (8–10 g/body FFM) for two days, and a nutritionally balanced reduction diet (RD), a typical diet containing 55% CHO, 15% protein, and 30% fat, on body composition and endurance performance in healthy young males [135]. Results revealed that both diets reduced body weight and body fat mass. However, while CKD-related weight loss is due to decreased body fat, body water, and lean body mass, RD leads to a reduction in body weight mainly by reducing body fat mass [135]. Among all of these findings, one should note that adherence to a weight loss diet is major factor in achieving a target that does not significantly require KD consumption.

Several studies determined the potential impact of HFD on aerobic capacity [16,17, 20,23,25,28,39]. It is well known that VO2max is referred to as a gold standard method to measure aerobic fitness [136]. Therefore, studies on KD, N-KD, and acute KE ingestion in endurance athletes stated that these diet manipulations had no effect on VO2max performance [16,17,20,23,25,39], except for a 4 week KD study on off-road cyclists by Zajac et al. [28]. Studies arguing that HFD was ineffective on aerobic capacity also showed that this HFD caused a decrease [16,23] or no change [25,39] in TT performance, and no alteration in time-to-exhaustion (TTE) performance [20]. Therefore, HFD seemed to fail to increase aerobic capacity and endurance in endurance athletes.

Researchers evaluated multiple performance-related factors such as TT performance [14, 19,23,25,29,33–36,38,39,41–43,45,46,48–50], TTE performance [18,20,21,26,31,37,51], lactate concentration during exercise [33,34,36,39], and post-exercise muscle damage [28] to determine the effects of HFD on sports performance. While research on TT and TTE performance in endurance athletes revealed controversial results, the majority of the studies declared that no alterations were observed in TT [14,19,25,29,34,36,38,39,41–43,45,49,50] and TTE [18,20,26,31,37] performance after the HF-associated applications. Additionally, two well-controlled studies of Burke et al. [16,23] underlined that a 3.5 week K-LCHF diet not only decreased 10 km race walk performance, but also increased oxygen cost and perceived exertion throughout exercise. These findings sugges<sup>t</sup> that HFD has no advantage or may even negatively affect exercise performance. However, some points should be taken into account when interpreting these findings. Five of eight studies on perceived fatigue during endurance performance revealed that no differences were detected between HFD and control trials [18,19,38,39,49]. Similar results were also observed in studies on lactate concentration during exercise [34,36,39]. It seems that HFD altered neither perceived exertion nor plasma lactate concentrations. Another important point for endurance performance is the maintenance of blood glucose concentration during exercise [38]. Changes in blood glucose levels during exercise were investigated in acute KB ingestion trials [34,36,38,39]. Three of four studies indicated that blood glucose concentrations were maintained during endurance exercise and were found to be similar between control groups [34,38,39].

Although these results are promising, blood glucose changes should also be examined in studies involving HFD manipulations. Additionally, a crossover study evaluating the efficiency of a 4 week NK-LCHF diet application on off-road cyclists stated that blood CK and LDH concentration, known as muscle damage biomarkers, significantly decreased at rest and during the 105 min exercise protocol in the NK-LCHF diet trial [28]. These findings also appear promising. It should be noted that studies reporting that TT or TTE performance did not change after HFD interpreted the study results based on statistical significance. It should also be noted that, although considered as statistically insignificant, a few minutes can be crucial in winning a race. Therefore, this point should be considered when interpreting the study results. Lastly, for post-exercise recovery, Volek et al. [24] indicated that long-term (at least 6 months) LCHF diets resulted in an increased fat oxidation rate and a higher peak exercise intensity in endurance athletes compared to counterparts consuming high-CHO low-fat diets. Moreover, although the LCHF diet group consumed 10% CHO, whereas the habitual high-CHO group consumed 59% CHO, there was no difference between the LCHF and high-CHO low-fat diets for 2 h post-exercise recovery [24]. These results sugges<sup>t</sup> that long-term LCHF diets can improve post-exercise recovery, especially in ultra-endurance events where the glycogen-sparing effect and adequate post-exercise recovery are crucial for a better performance. Keeping all these findings in mind, although studies on TT and TTE performance mostly found no advantages of HFD or revealed controversial results, performance-related parameters may be positively affecting the HFD. More work is required to clarify this information.

Ketone body consumption in endurance athletes may increase endurance performance by up-regulating physiological parameters and increasing metabolic efficiency [126]. For instance, Cox et al. [33] conducted comprehensive research including five separate studies on the effect of ketone esters (KE) on the performance of 39 endurance athletes. Twenty minutes after consumption of the ketone ester-based drink, blood ketone concentrations rapidly increased to 2 mmol/L and remained high with a slight drop, reaching a new steady state approximately 30 min following subsequent exercise at 75% Wmax exercise intensity. Findings from the study showed that acute nutritional ketosis caused by the consumption of KE resulted in metabolic improvements in endurance performance by enhancing metabolic flexibility and energy efficiency, rapidly altering substrate utilization towards ketone bodies for oxidative respiration, sparing intramuscular BCAA concentration by reducing BCAA deamination, increasing muscle fat oxidation even though in the

presence of glycogen, and decreasing blood lactate levels during exercise [33]. On the other hand, most of the studies (6 of 10) applying acute KB intake showed that this practice did not improve TT [34,36,38,39] and TTE performance [31,37]. Study findings remain unclear, and the impact of KB on exercise performance needs further clarification.

The efficacy of HFD on cognitive performance has been investigated in studies on acute KE [39] and KS [38] administration and fat-enriched feeding during high-CHO diet administration [50]. Prins et al. [38] administered one (22.1 g) or two (44.2 g) servings of KS or placebo to recreational male distance runners 60 min before a 5 km TT performance, and noted a possible dose–response interaction between KS supplementation and cognitive performance. On the other hand, studies including acute KE administration [39] and fat-enriched feeding during high-CHO diet administration [50] showed no alteration in cognitive performance. A study applying the high-CHO diet supplemented with fat on trained male cyclists highlighted that a possible explanation for this result is that the study protocol, including 1 h of fixed-task simulated TT performance, may not be sufficient to create mental fatigue [50]. However, the study on acute KE intake found similar results despite applying an exercise protocol (1 h submaximal exercise at 65% VO2max followed by a 10 km TT) that caused more fatigue [39]. Taken together, studies did not confirm the exact efficiency of HFD on cognitive performance and the interaction needs further investigation.

Few studies investigated the potential influence of HFD on immunologic and hormonal response in endurance athletes [15,22,30]. Assessing the impact of acute (2 day) and prolonged (2 week) adherence to an K-LCHF diet on exercise-induced cortisol, serum immunoglobulin A (s-IgA) responses in a randomized, crossover manner, researchers indicated that a lower cortisol response at week 2 was observed compared to day 2 in the K-LCHF trial (669 ± 243 nmol/L vs. 822 ± 215 nmol/L, respectively) [15]. However, a better exercise-induced cortisol response was found in the HCF trial at both day 2 and week 2 (609 ± 208 nmol/L and 555 ± 173 nmol/L, respectively). Additionally, no differences in s-IgA concentrations were observed at week 2 between the K-LCHF diet and high-CHO diet [15]. Another study by Shaw et al. [30] determined the impact of acute KE supplementation (R,S-1,3-butanediol (BD); 2×0.35 mg·kg−<sup>1</sup> BW; 30 min before and 60 min after exercise) on the T-cell-associated cytokine gene expression within stimulated peripheral blood mononuclear cells (PBMC) following prolonged, strenuous exercise in trained male cyclists. No alteration was detected in serum cortisol, total leukocyte and lymphocyte, and T-cell subset levels, IL-4 and IL-10 mRNA expression, and the IFN-γ/IL-4 mRNA expression ratio between the KE and placebo trials during exercise and recovery. However, a transient increase was observed in T-cell-related IFN-γ mRNA expression throughout exercise and recovery in the KE trial. Results indicated that acute KE supplementation may provide enhanced type-I T-cell immunity at the gene level [30]. The same researchers investigated the potential effect of a 4.5 week K-LCHF diet on resting and post-exercise immune biomarkers in endurance-trained male athletes in a randomized, repeated-measures, crossover manner [22]. T-cell-related IFN-γ mRNA expression and the IFN-γ/IL-4 mRNA expression ratio within multiantigen-stimulated PBMCs were greater in the K-LCHF trial compared to the high-CHO trial. Furthermore, a significant rise was observed in the multiantigen-stimulated whole-blood IL-10 production, an anti-inflammatory cytokine, post-exercise in the K-LCHF trial. The results indicated that a 4.5 week K-LCHF diet caused an increase in both pro- and anti-inflammatory T-cell-related cytokine response to a multiantigen in vitro [22]. Keeping the studies on immunologic and hormonal response to HFD in mind, although post-exercise pro- and anti-inflammatory T-cell-related cytokine response alters after a K-LCHF diet or acute KE supplementation, it remains uncertain how these alterations influence the immunoregulatory response. Therefore, more work is required to elucidate the interaction by adding clinical illness follow-up and tracking immunomodulatory metabolites using metabolomic approaches.

Antioxidant specialties of HFD may be discussed on the basis of KB [124]. Antioxidant activity of KBs is one of the multidimensional properties that determine their metabolic activity in the body. The main potential antioxidant properties of KB are mainly explained by its effects on neuroprotection, inhibiting lipid peroxidation and protein oxidation, and improving mitochondrial respiration [137]. However, as there is no study investigating the impact of KB on exercise-induced oxidative stress in endurance athletes and the evidence on the impact of KB on exercise-induced oxidative stress is limited, future studies in this field are needed.

Another therapeutic benefit of KD may be linked to increased Fibroblast Growth Factor 21 (FGF21) [132]. Fibroblast Growth Factor 21 acts as the primary regulator of skeletal muscle keto-adaptation by increasing activation of the AMP-activated protein kinase (AMPK)—sirtuins 1 (SIRT1)—peroxisome proliferator-activated receptor coactivator 1 (PGC-1) pathway, resulting in increased mitochondrial biogenesis, development of IMTGs, and ketolytic gene expression [138]. However, in a study on 5-d fat adaptation followed by 1-d CHO restoration, a significant decrease was observed in the exercise-induced AMPK-1 and AMPK-2 activity in the fat-adapted trial despite the higher AMPK-1 and AMPK-2 activity before exercise. Therefore, more work is required to interpret the possible interaction accurately.

Ketone bodies may have a particular metabolic advantage, not only providing a source of oxidizable carbon to maintain energy needs but also acting as a potential regulator of overtraining by directly regulating autonomic neural output and inflammation [139,140]. One study applying three weeks of KE intake during prolonged extreme endurance training investigated the effects of KE on overreaching symptoms [130]. Ketone ester ingestion significantly increased sustainable training load (15% higher than the control group), and prevented the increase in nocturnal adrenaline and noradrenaline excretion induced by strenuous training [130]. These findings sugges<sup>t</sup> that KE supplementation during exercise substantially reduces the development of overreaching, which is a detrimental factor for endurance performance. In addition, growth differentiation factor (GDF-15), an established biomarker for nutritional and cellular stress, increased 2-fold less in the KE group than the control group. However, this study was conducted on healthy, physically active males, and it is not exactly known whether the same effects can be achieved in endurance athletes [130]. For this reason, it is necessary to examine the same mechanism, especially on endurance athletes with intense and frequent training periods.

#### 3.2.2. Potential Risks Regarding High-Fat Diets

Some researchers have also investigated HFD's potential risks on endurance, including an increased oxygen cost and an impaired running economy [16,23], an altered blood acidbase status [17,31], compromised gastrointestinal (GI) symptoms [32,34,35,37,48], reduced bone formation markers [40], increased cholesterol and lipoprotein levels [27], a decreased appetite [37], and thereby worsened performance.

The deterioration of the running economy and increased oxygen cost during endurance exercise are considered to be major potential disadvantages of HFD. Burke et al. [16,23] demonstrated with two separate studies in elite race-walkers that a 3 week K-LCHF diet during intensity training impaired endurance performance by decreasing exercise economy, which has vital importance in endurance performance, despite enhancing peak aerobic capacity (VO2peak). Another study by Burke et al. claimed that although KD elevated glycogen availability, it still impaired endurance performance mainly by blunting the CHO oxidation rate [141]. In addition, LCHF diets can also impair endurance performance by increasing perceived fatigue [15,16,23]. The reason why K- LCHF diets cause increased fatigue is thought to be a gradual increase in non-esterified fatty acids (NEFAs) with the LCHF diet [142]. Non-esterified fatty acids compete with the tryptophan, a neurotransmitter highly associated with the central fatigue, for binding to albumin, thus resulting in an increase in free tryptophan transfer from the blood–brain barrier towards the brain. However, as we discussed above, the majority of studies found no alteration in perceived exertion during endurance performance [18,19,38,39,49].

Studies on well-trained endurance athletes revealed that neither keto-adaptation nor CHO restoration followed by keto-adaptation improves endurance performance, especially at multistage ultra-endurance events with intermittent sprints [42,45]. For instance, investigating the impact of a 6 days high-fat (68% fat) diet followed by 1 day CHO loading or high-CHO diet (68% CHO) for seven days on performance parameters during the 100 km time trial, Havemann et al. [45] found that 100 km time trial performance assessed by heart rate, perceived exertion, and muscle recruitment did not differ between groups; however, the 1-km sprint power output decreased more in the high-fat diet group than in high-CHO counterparts. Although an improvement was expected in high-intensity sprint bouts after an NK-LCHF diet due to its sparing effect on muscle glycogen, the findings revealed the opposite, decreasing the high-intensity sprint performance, a crucial parameter for endurance performance [45]. On the contrary, McSwiney et al. [14] also evaluated the impact of K-LCHF diets on 100 km TT performance and 6 s sprint peak power, indicating that although TT performance did not differ between the K-LCHF diet and high-CHO diet groups, 6 s sprint peak power significantly increased (+0.8 <sup>W</sup>·kg−<sup>1</sup> rise) compared to the high-CHO group ( −0.7 <sup>W</sup>·kg−<sup>1</sup> decrease). More research is required to clarify these contradictory results.

Maintaining the acid-base balance in the body during exercise, especially during strenuous exercise, is important to delay acidosis and fatigue and thus to maintain endurance performance [143]. Exercise is a well-known factor that alters the acid-base state [143]. In addition to exercise, the macronutrient composition of dietary patterns can also affect acid-base balance and systemic pH and HCO3 levels [31]. Some researchers claimed that HFD can alter circulating acidity by increasing acidic KB circulation in the body [144], while others state that acid-base balance can be well regulated by improving the adaptive mechanisms, regardless of diet [17]. The potential effect of HFD on blood acid-base status, blood pH, and HCO3 concentrations was evaluated in only two studies of endurance athletes. The potential effect of HFD on blood acid-base status, blood pH, and HCO3 concentrations was evaluated in two studies, one evaluating a 3 week ketogenic diet [17] and the other an acute KE intake in endurance athletes [31]. The study findings showed that neither K-LCHF diet nor acute KE intake affected blood pH and HCO3 status and acidbase status [17,31]. One explanation is that both studies included well-trained endurance athletes. It is suggested that well-trained athletes can regulate the body acid-base balance well regardless of the diet by developing a metabolic adaptation to strenuous exercise. Therefore, the potential effect of HFD on acid-base status can be interpreted as negligible when applied to well-trained endurance athletes.

Gastrointestinal symptoms triggered by an HFD have commonly been seen during KB consumption [32,34,35,37]. A study investigating the kinetics, safety and tolerability of KB revealed that ketone esters may only cause GI symptoms when high doses (2.1 <sup>g</sup>·kg−1) are consumed [145]. However, although studies administered a low-dose KE in endurance athletes, the findings stated that acute KE ingestion caused an increase in low to severe GI symptoms, including nausea, reflux, dizziness, euphoria, and upper-abdominal discomfort [32,34,35,37]. One study by Dearlove et al. [32] compared the dose–response interaction between acute low- or high-dose KE ingestion (0.252 <sup>g</sup>·kg−<sup>1</sup> vs. 0.75 <sup>g</sup>·kg−1, respectively) and GI symptoms. Findings showed that no GI discomfort was observed in the low-dose KE ingestion, while nausea symptoms were elevated in the high-dose KE trial. Although the high dose administered in this study (0.75 <sup>g</sup>·kg−1) remained much lower than the high dose (2.1 <sup>g</sup>·kg−1) that was claimed to cause GI symptoms, it still caused exercise-induced nausea in endurance athletes. In addition, Mujika [48] investigated the race performance and GI symptoms of a LOV male endurance athlete who adhered to an LCHF diet for 32 weeks. The athlete participated in three professional races while on the LCHF diet in weeks 21, 24, and 32. Although he suffered worse race experiences on the LCHF diet, no alteration was observed in GI symptoms. This result may be due to the athlete's adaptation to the ketogenic diet [48]. Taken together, while long-term keto-adaptation may inhibit the increase in GI symptoms, it should be taken into account when applying to endurance

athletes that acute KE intake may be disadvantageous on exercise-induced GI symptoms. Interestingly, Zinn et al. [21] showed that endurance athletes suffered from constipation during the diet application after a 10 week K-LCHF diet, which might be important for the gu<sup>t</sup> microbiome and well-being. This possibility may also be kept in mind while applying a ketogenic diet. In case of a similar situation, fiber and water intake should be calculated and closely monitored to eliminate constipation-associated problems.

Another less-studied potential disadvantage of HFD is its potential impact on decreasing appetite [37], bone formation markers [40], and increasing cholesterol and lipoprotein profile [27]. A randomized, crossover study evaluating the effects of acute KE ingestion early in a cycling race on glycogen degradation in highly trained cyclists showed a significant attenuation in the perception of hunger, determined using a validated 10-point visual analog scale [37]. This potential effect of HFD on appetite should be taken into account, especially during HFD administration planned for long-term application.

Heikura et al. [40] investigated the effects of a 3.5 week K-LCHF diet followed by CHO restoration on bone biomarkers in male and female race walkers. Their findings showed a meaningful increase in bone resorption markers at rest and post-exercise while a significant attenuation in bone formation markers at rest and throughout exercise in K-LCHF diet trial occurred. However, these alterations partially recovered after CHO restoration [40]. As only one study investigated the interaction between bone markers and ketogenic diets in endurance athletes, and a recent narrative review on ketogenic diets and bone health noted that we do not have enough high-quality experimental research to adequately clarify the potential disadvantages of ketogenic diets on bone health, we need more high-quality research on this topic.

Only one cross-sectional study of 20 competitive ultra-endurance athletes investigated the interaction between a long-term low-CHO diet and the circulating lipoprotein and cholesterol profiles [27]. Although a higher level of exercise tended to lower total and LDL-C concentrations, a hypercholesterolemic profile was observed in ultra-endurance athletes who adhered to a low-CHO diet, suggesting that a possible explanation may involve an expansion of the endogenous cholesterol pool during keto-adaptation and may remain higher on a low-CHO diet. Further, a higher consumption of saturated fat (86 vs. 21 <sup>g</sup>·day−1) and cholesterol (844 vs. 251 mg·day−1), and lower fiber intake (23 vs. 57 <sup>g</sup>·day−1) may be another cause of these hypercholesterolemic profiles of ultra-endurance athletes [27]. However, due to the small sample size (*n* = 20) and the lack of checking for familial hypercholesterolemia or specific polymorphisms [27], future work is needed to evaluate this interaction in depth.

Another possible pathway is that KD high in protein causes an increase in ammonia, thereby altering both brain energy metabolism and neuronal pathways, thus triggering central fatigue [146]. Both NEFA and ammonia may lead to increased central fatigue during exercise in endurance athletes adopting KD [142]. The interaction between the gut–brain axis can have critical importance to reveal performance- and, especially, fatiguerelated metabolism during endurance events [147]. However, none of the HFD studies on endurance athletes studied the gut–brain axis, increased ammonia concentration, or endurance performance. Another point regarding a high protein intake during KD is that a high protein consumption can disrupt ketosis by providing gluconeogenic precursors, thus inducing gluconeogenesis [148]. Therefore, moderate protein consumption is generally recommended during KDs. As we know that endurance athletes tend to consume more protein intake (1.2–2.0 <sup>g</sup>·kg−<sup>1</sup> BW·day−1) [149], this important effect of protein on ketosis should be kept in mind during the KD administration periods.

There are some important points that need to be considered before applying an HFD in endurance athletes. During NK-LCHF diet applications, the metabolic adaptation of muscle may evolve towards oxidation of fat as the primary energy source (maximum fat oxidation rate (fat max) from 0.4–0.6 <sup>g</sup>·min−<sup>1</sup> to 1.2–1.3 <sup>g</sup>·min−1) [139]. However, glycogen stores may not provide enough glucose to power the brain, thus increasing fatigue [150] and decreasing endurance performance. For this reason, the adaptation period should be chosen carefully in order to alleviate the side effects of transition periods. Phinney et al. [20] noted that ketogenic high-fat diets may impair performance at first (a reduction of approximately 20%), but improvements in performance (up to a 155% increase) can be observed after metabolic adaptation to the ketogenic state.

Another important point that needs to be considered while planning further studies on HFD is to evaluate blood ketone concentration at frequent intervals during the study application period [151]. A review investigating the role of ketone bodies on physical performance found that 7 out of 10 studies included in the review failed to reach BOHB concentrations at the 2 mmol/L threshold, but only caused an acute ketosis state (B-OHB > 0.5 mmol/L) [151]. Another significant point is which KB type should be used [152]. The impact of ketone bodies on metabolism differs according to the type (ester-based form or salt-based form), and optical isoform (e.g., L or D isoforms of BOHB) consumed [137]. For example, D-βOHB is produced from acetoacetate (AcAc), released by the liver, and is actively used in metabolic pathways [153], while L-OHB is an intracellular metabolite known for having less activity in oxidative metabolism [150]. Therefore, L-βOHB supplementation may not provide the performance-related benefits of ketone bodies. These results explain that the specific effect of KD or KB on physical performance awaits further investigation, as most studies of KB failed to achieve the required ketone concentrations or applied ineffective KB to enhance endurance performance [152].

To conclude, there are several HFD strategies, as discussed in detail above, practiced by endurance athletes. However, while these diets may provide performance and health benefits, they are sometimes not effective at all or create many problems for endurance athletes. In addition, the physiological response to acute (exogenous) or endogenous nutritional ketosis may vary between highly trained endurance athletes and untrained individuals [140]. Therefore, it should be noted that these strategies may not be suitable for all endurance athletes. At first glance, while high-fat diets may seem like a promising approach to endurance performance, more research is needed to keep in mind all study results.
