The Effects of Carbohydrate versus Fat Restriction on Lipid Profiles in Highly Trained, Recreational Distance Runners: A Randomized, Cross-Over Trial

growing number of endurance athletes have considered switching from a traditional high-carbohydrate/low-fat (HCLF) to a low-carbohydrate/high-fat (LCHF) eating pattern for health and performance reasons. However, few studies have examined how LCHF diets affect blood lipid pro les in highly-trained runners. In a randomized and counterbalanced, cross-over design, athletes (n= 7 men; VO 2max:

parker.hyde@ung.edu *Correspondence: pjprins@gcc.edu; Tel.: +1-724-458-3863 Abstract: A growing number of endurance athletes have considered switching from a traditional high-carbohydrate/low-fat (HCLF) to a low-carbohydrate/high-fat (LCHF) eating pattern for health and performance reasons. However, few studies have examined how LCHF diets affect blood lipid pro les in highly-trained runners. In a randomized and counterbalanced, cross-over design, athletes (n= 7 men; VO 2max: 61.9 6.1 mL/kg/min) completed six weeks of two, ad libitum, LCHF (6/69/25% en carbohydrate/fat/protein) and HCLF (57/28/15% en carbohydrate/fat/protein) diets, separated by a two-week washout. Plasma was collected on days 4, 14, 28, and 42 during each condition and analyzed for: triglycerides (TG), LDL-C, HDL-C, total cholesterol (TC), VLDL, fasting glucose, and glycated hemoglobin (HbA1c). Capillary blood beta-hydroxybutyrate (BHB) was monitored during LCHF as a measure of ketosis. LCHF lowered plasma TG, VLDL, and TG/HDL-C (allp< 0.01). LCHF increased plasma TC, LDL-C, HDL-C, and TC/HDL-C (allp< 0.05). Plasma glucose and HbA1c were unaffected. Capillary BHB was modestly elevated throughout the LCHF condition (0.5 0.05 mmol/L). Healthy, well-trained, normocholesterolemic runners consuming a LCHF diet demonstrated elevated circulating LDL-C and HDL-C concentrations, while concomitantly decreasing TG, VLDL, and TG/HDL-C ratio. The underlying mechanisms and implications of these adaptive responses in cholesterol should be explored. Keywords:low-carbohydrate; low-fat; crossover; endurance athletes; cholesterol; lipid pro les 1. Introduction Endurance athletes are encouraged to consume a carbohydrate-rich diet (7–12 g/kg/day) to achieve peak performance [1,2]. Carbohydrate-restriction (<50 g/day) has been adopted in recent years by an increasing number of athletes who want to test its putative roles in training, health, weight loss, body composition, gastrointestinal tolerance, and recovery [3,4]. The diet relies on the main premises that (1) an increase fat oxidation to a threshold where lipids become the predominant fuel during prolonged, submaximal exertion (~65% VO2max) confers a substrate advantage that does not need refueling [5–7] and (2) maintaining this elevated fat Nutrients2022,14, 1135.

Nutrients2022,14, 1135 2 of 14 oxidation rate may delay glycogen degradation for race stages where high-intensity work (>70% VO2max) output is required (i.e., sprints) [ The evidence supporting low-carbohydrate/high-fat (LCHF) diets for aerobic perfor- mance is mixed, and whether one dietary strategy is best suited for athletic activities is still being extensively investigated. However, a well-documented effect when switching from a habitual diet to an LCHF diet is observed in blood lipids. Markers such as total cholesterol (TC), low/high-density lipoproteins (LDL/HDL) and triglycerides (TG), expressed either as absolute values or relative ratios, are measured to characterize cardiovascular risk. The goal of lifestyle interventions is to primarily lower TC, LDL, and TGs and increase HDL to induce a favorable lipid pro le [9,10], an effect that LCHF interventions have demon- strated in non-athletic populations undergoing weight-loss [11,12]. Studies that previously tested LCHF responses in athletes reported variable results, with some documenting a decrease in circulating lipids [13], whereas others reported neutral [14,15] or an increasing trend [16,17] across all lipid parameters. Additionally, there is strong evidence to suggest that aerobic exercise alone may suf ciently act as a positive modulator of serum lipids, primarily HDL, independent of other factors [18,19]. Despite the uses of LCHF in a range of sports [20–22], few studies have carefully examined how diet affects lipid pro les in endurance athletes [23]. In a cross-sectional study of ultra-endurance runners, the group habitually consuming a LCHF diet (>20 months;n= 11) demonstrated greater circulating cholesterol, both as total, large diameter LDL, and HDL, compared to the high-CHO comparison group [24,25]. Lambert et al. conducted a small crossover study in cyclists (n= 5) and showed that LCHF did not signi cantly alter blood lipids whilst preserving the cycling performance compared to a habitual mixed diet [26]. Lastly, O'Neal et al. revealed that, in well-trained middle-aged runners, three weeks of a mixed diet crossed over to LCHF (n= 8), raised TC and LDL concentrations (~30 and 20 mg/dL increase, respectively), but not HDL, while TG decreased signi cantly, independent of diet [27]. These ndings suggest that blood lipid pro les in

to a habitual mixed diet [26]. Lastly, O'Neal et al. revealed that, in well-trained middle-aged runners, three weeks of a mixed diet crossed over to LCHF (n= 8), raised TC and LDL concentrations (~30 and 20 mg/dL increase, respectively), but not HDL, while TG decreased signi cantly, independent of diet [27]. These ndings suggest that blood lipid pro les in endurance populations may vary in response to both diet and exercise (i.e., LDL/HDL increase; TG decrease). The most plausible explanation for between-group variability is methodology differences across studies. Our previously published cross-over approach [15] was intended to evaluate both within- and between-group changes and has been recently recommended in a LCHF diet and performance review paper [23] as a model for future studies that plan to evaluate diet-dependent effects in athletes. The main goal of this exploratory study was to determine the effects of LCHF or HCLF feeding on fasted blood lipids and glucose markers in long distance runners. To answer this question, we performed the rst randomized and balanced, cross-over trial in highly trained endurance athletes (n= 7 men). Each athlete was guided on how to implement LCHF and HCLF lifestyles ad libitum for six weeks, separated by a two-week washout between experimental conditions. Dietary compliance during the LCHF condition was evaluated using food logs and tracking the primary circulating ketone body in capillary blood (beta-hydroxybutyrate; BHB). Plasma was analyzed for total cholesterol (TC), very- low/low/high-density lipoproteins (VLDL, LDL-C, HDL-C), triglycerides (TG), glucose and glycated hemoglobin (HbA1C). 2. Materials and Methods 2.1. Experimental Design and Participants This was an exploratory, randomized, counterbalanced crossover-trial study design. The main goal was to evaluate the blood lipid responses in highly trained, recreational endurance athletes who were motivated to undergo 12 weeks of experimental, dietary inter- vention phases concurrent with their training regimen. Inclusion criteria were divided into three objective categories: running performance (<21 0 00” 5-km within 3 months of study enrollment; >32 km of running per week; >2 years of running experience); age (18–45 years); habitual dietary intake (>50% total energy needs from carbohydrates). Exclusion criteria included hypercholesterolemia (>200 mg/dL), hypertriglyceridemia

experimental, dietary inter- vention phases concurrent with their training regimen. Inclusion criteria were divided into three objective categories: running performance (<21 0 00” 5-km within 3 months of study enrollment; >32 km of running per week; >2 years of running experience); age (18–45 years); habitual dietary intake (>50% total energy needs from carbohydrates). Exclusion criteria included hypercholesterolemia (>200 mg/dL), hypertriglyceridemia (>150 mg/dL), ha-

Nutrients2022,14, 1135 3 of 14 bitually consuming a ketogenic or low-carbohydrate diet (<20% total energy needs from carbohydrates) or being prescribed lipid- or glucose-lowering medication. Before enrolling in the study, participants were fully informed of any associated risks and discomforts prior to giving their written informed consent to participate. The experimental protocol was approved by the Grove City College Institutional Review Board prior to implementation. Participants (n= 7 men) who met the criteria were invited to in-person consent visit where the protocol and study responsibilities were described in greater detail. Each participant received a unique study identi cation number to protect their real identity. The experimental order was established by codifying each participant (1–7) and dietary conditions (A = LCHF; B = HCLF) into a randomized, online number generator (www.randomizer.org; accessed on 19 June 2018). After randomization, each participant was assigned to an ad libitum, low-carbohydrate/high-fat diet (LCHF) or a high- carbohydrate/low-fat diet (HCLF) for six weeks. A two-week, also ad libitum, mixed diet (i.e., CHO > 50 g/day and >20% fat) washout stage separated the two experimental dietary phases. Participants reported to the testing site bi-weekly for data collection. Baseline characteristics (Table) and a graphical summary of experimental design are presented below (Figure).Nutrients 2021, 14, x FOR PEER REVIEW 3 of 14 intervention phases concurrent with their training regimen. Inclusion criteria were di- vided into three objective categories: running performance (<21′00″ 5-km within 3 months of study enrollment; >32 km of running per week; >2 years of running experience); age (18–45 years); habitual dietary intake (>50% total energy needs from carbohydrates). Ex- clusion criteria included hypercholesterolemia (>200 mg/dL), hypertriglyceridemia (>150 mg/dL), habitually consuming a ketogenic or low-carbohydrate diet (<20% total energy needs from carbohydrates) or being prescribed lipid- or glucose-lowering medication. Before enrolling in the study, participants were fully informed of any associated risks and discomforts prior to giving their written informed consent to participate. The experi- mental protocol was approved by the Grove City College Institutional Review Board prior to implementation. Participants (n = 7 men) who met the criteria were invited to in-person consent visit where the

glucose-lowering medication. Before enrolling in the study, participants were fully informed of any associated risks and discomforts prior to giving their written informed consent to participate. The experi- mental protocol was approved by the Grove City College Institutional Review Board prior to implementation. Participants (n = 7 men) who met the criteria were invited to in-person consent visit where the protocol and study responsibilities were described in greater de- tail. Each participant received a unique study identification number to protect their real identity. The experimental order was established by codifying each participant (1–7) and dietary conditions (A = LCHF; B = HCLF) into a randomized, online number generator (www.randomizer.org; Retrieved June 19, 2018). After randomization, each participant was assigned to an ad libitum, low-carbohydrate/high-fat diet (LCHF) or a high-carbohy- drate/low-fat diet (HCLF) for six weeks. A two-week, also ad libitum, mixed diet (i.e., CHO > 50 g/day and >20% fat) washout stage separated the two experimental dietary phases. Participants reported to the testing site bi-weekly for data collection. Baseline characteristics (Table 1) and a graphical summary of experimental design are presented below (Figure 1). Figure 1. Experimental Approach. In a randomized, counterbalanced, cross-over manner, seven rec- reationally trained endurance athletes were assigned to consume either a low-carbohydrate/high- fat diet (LCHF) or high-carbohydrate/low-fat diet (HCLF) for six weeks. A two-week washout was allowed between each dietary phase. All participants maintained a constant exercise regimen and a 3-day food log throughout the experimental phases. Repeat intravenous and capillary blood measures were collected on days 4, 14, 28, and 42 during each feeding condition. The exploratory goal of this study was to assess the main effects and interactions of LCHF and HCLF on compre- hensive blood lipid and glucose panel markers. This figure was created with BioRender ® . Figure 1. Experimental Approach. In a randomized, counterbalanced, cross-over manner, seven recreationally trained endurance athletes were assigned to consume either a low-carbohydrate/high- fat diet (LCHF) or high-carbohydrate/low-fat diet (HCLF) for six weeks. A two-week washout was allowed between each dietary phase. All participants maintained a constant exercise regimen and a 3-day food log throughout

was created with BioRender ® . Figure 1. Experimental Approach. In a randomized, counterbalanced, cross-over manner, seven recreationally trained endurance athletes were assigned to consume either a low-carbohydrate/high- fat diet (LCHF) or high-carbohydrate/low-fat diet (HCLF) for six weeks. A two-week washout was allowed between each dietary phase. All participants maintained a constant exercise regimen and a 3-day food log throughout the experimental phases. Repeat intravenous and capillary blood measures were collected on days 4, 14, 28, and 42 during each feeding condition. The exploratory goal of this study was to assess the main effects and interactions of LCHF and HCLF on comprehensive blood lipid and glucose panel markers. This gure was created with BioRender ® .

Nutrients2022,14, 1135 4 of 14 Table 1.Subject Characteristics (n= 7). Variable Mean SD Age (years) 35.6 8.4 Height (cm) 178.7 4.1 Weight (kg) 68.6 1.6 BMI (kg/m 2 ) 21.5 1.1 Body Fat (%) * 5.0 1.3 Fat Mass (kg) 3.5 1.0 Lean Mass (kg) 65.1 1.5 VO 2max(mL/kg/min) 61.9 6.1 Running Distance Per Week (km) 63.0 27.1 Running Experience (years) 15.1 7.1 * Determined by bioelectrical impedance. VO2max, maximal oxygen consumption. 2.2. Nutrition and Exercise Guidelines A registered dietitian educated and guided each athlete a priori of experimental phases on how to implement LCHF and HCLF guidelines at home through direct counseling and handouts. The primary macronutrient targets for LCHF and HCLF were expressed both as percentage of total daily energy intake (% en) and per gram basis: LCHF: <50 g/day carbohydrate, 75–80% en fat, 15–20% en protein. HCLF: 60–65% en carbohydrate, 20% en fat, 15–20% en protein. Participants were explicitly instructed to consume the diets until they reach satiety. Consumption of a wide range of foods was encouraged to minimize micronutrient de - ciencies. To ensure that mineral status was met for LCHF, we recommended including an additional 1–2 g/day of iodized table salt to offset the additional loss of sodium as- sociated with a reduction in total carbohydrate intake [28]. Weekly energy intake and relative macronutrient distribution was monitored and estimated via 3-day weighed food records, capturing two consecutive weekdays and a weekend day. A digital scale (Ozeri ZK14-S Pronto, San Diego, CA, USA) calibrated to the nearest 0.1 g was provided to each athlete prior to experimental phases to improve food tracking accuracy (intended for both dry and cooked items). Dietary macro- and micronutrients were calculated by the same registered dietitian using advanced nutrient software (Nutritionist Pro, Axxya Systems, Redmond, WA, USA). In addition to food records, compliance to the LCHF dietary regimen was monitored by daily, morning capillary blood ketone measurements as determined by beta-hydroxybutyrate (BHB). To minimize confounding exercise effects, participants were instructed to select and maintain a constant training intensity and volume that they could adhere to for 14 weeks. Subjects were instructed

Pro, Axxya Systems, Redmond, WA, USA). In addition to food records, compliance to the LCHF dietary regimen was monitored by daily, morning capillary blood ketone measurements as determined by beta-hydroxybutyrate (BHB). To minimize confounding exercise effects, participants were instructed to select and maintain a constant training intensity and volume that they could adhere to for 14 weeks. Subjects were instructed to record their training habits (mode/duration/intensity) one week prior to commencing experimental dietary phases. Post-hoc training load analysis revealed no differences in free-living exercise habits throughout the study. Detailed description of exercise testing during the study was reported previously [15]. 2.3. Laboratory Protocols The data presented in this manuscript was collected as part of a larger project [15] examining the effects of LCHF and HCLF on dietary and exercise adherence, running performance, physiological and metabolic adaptations, and change in body composition. Detailed description of general methods and other results have been previously detailed by Prins et al. [15]. In brief, a sequence of tests was performed on day 4, 14, 28, and 42 during each dietary phase. Participants reported to the testing laboratory (Grove City College Exercise Science Human Performance Laboratory) between 6:00 and 9:00 a.m. after an overnight fast (8–12 h). Upon arrival, hydration status was assessed in urine via speci c gravity (USG < 1.020) using a portable light refractometer (Reichert™, Buffalo, NY, USA). Athletes who did not meet this threshold were offered ~250 mL of water and re-tested for hydration.

Nutrients2022,14, 1135 5 of 14 Following an 8–12-h fast, a trained phlebotomist collected fasting venous blood sam- ples via venipuncture. Before sampling, participants were asked by a researcher if they had anything to eat or drink that morning. Venous samples were taken between 6 a.m. and 9 a.m. Samples were collected in a lithium heparin BD Vacutainer Plasma Tube and immediately placed on ice after collection. Whole blood was analyzed within 10 min of collection using Abaxis Piccolo Xpress point of care chemistry analyzer (Abaxis Inc., Union City, CA, USA) and Lipid Panel Plus discs assays for the measurement of total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), triglycerides (TG), very low-density lipoprotein (VLDL), TC to HDL-C ratio (TC/HDL-C), TG to HDL-C ratio (TG/HDL-C) and glucose. LDL-C was calculated automatically using the Friedwald equation [29]. Prior to blood analysis, calibration and quality control measures were taken by running control reagents, and all values fell within the acceptable device range. Glycated hemoglobin was analyzed using an Alere A nion HbA1c Analyzer (Alere Technologies). Fasted capillary BHB was measured by participants every morning via nger sticks (<100 L blood sample) using a commercially available glucometer tted for ketone reagent strips (Precision Xtra, Abbott Diabetes Care Inc., Almeda, CA, USA). Daily BHB values were retrieved on test days from the internal device memory and manually stored into a secure database by the laboratory team members. 2.4. Statistics Analyses were performed using SPSS ver. 25 (SPSS, Inc., Chicago, IL, USA). Two-taila signi cance was set atp< 0.05. To address our exploratory objective, we analyzed main effects and interactions between LCHF and HCLF using a 2 (condition) 4 (time) repeated measures ANOVA. Subject characteristics at baseline are presented as descriptive variables. All the variables of interest analyzed were screened for normality using Shapiro-Wilks test. Assumption of sphericity was con rmed using Mauchly's test; variables that violated sphericity were treated with the Greenhouse-Geiser correction. Bonferroni correction was applied for multiple post-hoc comparisons. All data is presented as mean SD. Figures were created with BioRender ® and graphs were designed in GraphPad

variables. All the variables of interest analyzed were screened for normality using Shapiro-Wilks test. Assumption of sphericity was con rmed using Mauchly's test; variables that violated sphericity were treated with the Greenhouse-Geiser correction. Bonferroni correction was applied for multiple post-hoc comparisons. All data is presented as mean SD. Figures were created with BioRender ® and graphs were designed in GraphPad Prism (GraphPad Software, Inc., San Diego, CA, USA; ver. 9.1). 3. Results Based on food records, participants adhered to the general principles of both diet guidelines (Table). There were no signi cant differences in average total energy intake between the two conditions (DLCHF HCLF = 110 kcal/day;p= 0.686). Participants met most macronutrient goals; during the LCHF experimental condition participants reported weekly carbohydrate intake below the <50 g/d threshold (43 6 g/day; 6% en), whereas average CHO intake during HCLF increased nearly ~10-fold (402 32 g/day; 56% en). Daily fasted capillary blood HB concentrations averaged 0.5 0.05 mmol/L throughout the six-week LCHF condition (Figure). A subset of participants met the nutritional ketosis threshold of BHB > 0.5 mmol/L (n= 2), whereas others met the borderline criteria (n= 1) or remained below this level (n= 4). Weekly BHB averages did not change signi cantly over time relative to the rst week of the LCHF condition (p= 0.286).

This study examines the effects of LCHF diets on blood lipid profiles in trained runners.