Continuous Protein Supplementation Reduces Acute Exercise-Induced Stress Markers in Athletes Performing Marathon

aim of this study was to determine the changes in endurance performance and metabolic, hormonal, and in ammatory markers induced by endurance stress (marathon race) in a combined strategy of training and dietary protein supplementation. The study was designed as a randomised controlled trial consisting of regular endurance training without and with a daily intake of a soy protein-based supplement over a three-month period in 2 15 (10 males and 5 females per group) endurance-trained adults. Body composition (body mass, BMI, and fat mass) was determined, and physical tness was measured by treadmill ergometry at baseline and after 3 months of intervention; changes in exercise-induced stress and in ammatory markers (CK, myoglobin, interleukin-6, cortisol, and leukocytes) were also determined before and after a marathon competition; eating behaviour was documented before and after intervention by a three-day diet diary. Although no signi cant in uence on endurance performance was observed, the protein supplementation regime reduced the exercise-induced muscle stress response. Furthermore, a protein intake of 20% of total energy intake led to a lower-level stress reaction after the marathon race. In conclusion, supplementary protein intake may in uence exercise-induced muscle stress reactions by changing cellular

a three-day diet diary. Although no signi cant in uence on endurance performance was observed, the protein supplementation regime reduced the exercise-induced muscle stress response. Furthermore, a protein intake of 20% of total energy intake led to a lower-level stress reaction after the marathon race. In conclusion, supplementary protein intake may in uence exercise-induced muscle stress reactions by changing cellular metabolism and in ammatory pathways. Keywords:protein-rich diet; endurance exercise; muscle stress; in ammation 1. Introduction It is well accepted that nutrition is a limiting factor in human performance and that athletes should ensure that they meet their daily energy needs to balance their requirements for energy expenditure [1]. Independent of total energy intake, the speci c timing and macronutrient composition of energy intake throughout the day has the potential to modulate the body's adaptation to exercise [2]. Furthermore, macronutrient composition should be adapted to the regularly performed training or sport modality since different sports have different energetic and nutritional requirements [3]. With respect to dietary protein, the supply of essential amino acids is critically linked to muscle protein synthesis and can promote skeletal muscle hypertrophy in response to chronic resistance training [4]. However, evidence linking protein intake to a bene t in endurance sports has not been clearly established [5]. Authors have suggested that protein intake during or immediately after exercise may induce an ergogenic effect on endurance performance, preferably in combination with the intake of carbohydrates as the rst-choice energy supply [2,6–8]. Nevertheless, there remains no consensus on whether the metabolic and systemic effects of proteins is simply a consequence of providing extra energy or rather, may be a speci c bene t regarding the protein source as well as to the intake of biological Nutrients2021,13, 2929.

Nutrients2021,13, 2929 2 of 10 active peptides, which are also increasingly absorbed via the increased or targeted supply of proteins [9–11]. Therefore, we performed a randomised controlled trial (RCT) involving a group of experienced athletes to evaluate the effects of a combination of endurance training and a regular intake of a protein-rich dietary supplement over a period of 3 months in preparation for a marathon race. These athletes also partook in a local marathon at the end of the intervention. The main aim of the study was to investigate whether the intake of the protein supplement in uences endurance performance and whether its supplementation reduces exercise-induced muscular or systemic stress reactions. 2. Materials and Methods 2.1. Study Design The study was designed as an experimental RCT to determine the effects of a protein- rich supplementation regime across a 3-month training period on the endurance perfor- mance and training adaptation, ultimately evaluated with a marathon race at the end of the intervention compared to a control group. Participants study visits were conducted at baseline (termed “a”) and at the end of the 3-month training intervention (termed “b”) as well as before (termed “c”) and 1 h after (termed “d”) the marathon race at the end of intervention. The period between visits “b” and “c” lasted approximately 7 days, but the athletes had to continue with the protein supplementation regimen until the marathon race day. After the “a" visit, study participants were randomly assigned either to the intervention or control group. 2.2. Participants After contacting and advertising the study protocol to regional sports clubs, 30 out of 52 applicants met the criteria for randomisation (de ned as experienced endurance runners) to either a verum (V;n= 15, 10 males, 5 females) or a control group (C;n= 15, 10 males, 5 females). Participants had to ful l the following inclusion criteria: to be clinically healthy endurance-trained adults, aged between 18–60 years, and a body mass index (BMI) between 18–25 kg/m 2 . Additional inclusion criteria were: constant roadwork training (ca. 3 times per week of 25–30 km endurance training), non-use of other nutritional or

control group (C;n= 15, 10 males, 5 females). Participants had to ful l the following inclusion criteria: to be clinically healthy endurance-trained adults, aged between 18–60 years, and a body mass index (BMI) between 18–25 kg/m 2 . Additional inclusion criteria were: constant roadwork training (ca. 3 times per week of 25–30 km endurance training), non-use of other nutritional or ergogenic supplements, no blood lipid- or glucose-lowering therapy within the preceding 12 months, being non-smoker, alcohol consumption <40 g/d, and a relative maximal oxygen consumption (VO2max) between 45–65 mL/kg/min. The study protocol was registered and approved by the Ethical Commission of the University of Freiburg (reg. no. 231/12). The study was conducted in accordance with the ethical standards established in the 1964 Declaration of Helsinki and its later amendments [12]. All participants were informed verbally and supported by a written participant information sheet, and signed consent to participate in the study was obtained. 2.3. Protein Supplementation The supplement used (comparable with the commercially available product Almased- Vitalkost ® ; Almased-Wellness-GmbH, Bienenbüttel, Germany) is composed of soy protein, skimmed milk powder, and honey without additives. The protein content of this product is 53.3% (83% soy-protein-isolate, and 17% milk protein). Given the special marathon conditions, each 50 g portion was supplemented with 9 g palatinose, 30 mg carnitine, 10 mg coenzyme Q10, and 0.5 mg -lipoic acid. One portion thereby equated to 60 g of powder dissolved in 300 mL water, providing 217 kcal, 27.2 g protein, 24.6 g CHO, and 1.0 g fat. Participants were instructed to drink one portion twice per day, in the morning before breakfast and in the afternoon or evening, with each intake always approximately at the same time of day and with a delay before ingestion of other meals to account for the low glycaemic load of the product.

Nutrients2021,13, 2929 3 of 10 2.4. Measures The participants' accordance with the inclusion criteria was con rmed in the initial comprehensive health screening before the intervention. Laboratory analysis of blood samples as well as the measurement of anthropometric parameters were concomitantly performed. Anthropometry comprised body height (BH), body mass (BM), and body mass index (BMI). BM was measured in light clothing to the nearest 0.1 kg, BH to the nearest 0.5 cm (Seca Stadiometer 274, Seca GmbH & Co. KG, Hamburg, Germany), and BMI was calculated with the formula: weight in kilogram divided by height in meters squared. Percentage body fat (FM%) was calculated using the Siri equation [13] with the log sum of skinfold thickness (SFT) at 4 measuring sides (triceps, biceps, subscapular, and suprailia). The SFT was measured using a skinfold calliper (Lange Skinfold Caliper, Beta Technology Inc., Noblesville, IN, USA). The laboratory analyses included selected metabolic variables for performance diagnostics (blood lactate concentration [14]), whole blood cell counts (including leukocytes [15]), selected blood indicators of muscle damage and systemic stress (creatine kinase (CK) [16], myoglobin [17] and interleukin-6 (IL-6) [18]), and the hormone cortisol [19]. Biochemical blood variables were determined by venous blood sampling. Leukocyte count, cortisol, and indicators of muscle damage and stress response were utilised in the calculation of a “stress score” (unpublished “in-house” method to determine overall exercise-induced stress response), which was expected to be noticeably increased after bouts of hard endurance exercise, such as a marathon race. For this purpose, the participants' values for each of the variables were ranked in ascending order and categorised into three equally sized groups. For each parameter, the participants were assigned to one of the three categories, reaching a stress score of 1 for a relatively low parameter increase, 2 for a moderate increase, and 3 for a high increase. Hence, for each participant, a nal total stress score was calculated ranging from a possible minimum of 5, showing low levels for all 5 variables, to a maximum score of 15. Mean stress scores were calculated for both study groups. The comprehensive health screening also included performance

parameter increase, 2 for a moderate increase, and 3 for a high increase. Hence, for each participant, a nal total stress score was calculated ranging from a possible minimum of 5, showing low levels for all 5 variables, to a maximum score of 15. Mean stress scores were calculated for both study groups. The comprehensive health screening also included performance diagnostics to assess the training status of the participants and were conducted as a treadmill test using spiroergometric equipment (ZAN Messgeräte GmbH, Oberthulba, Germany) following the ZAN protocol of sequential 3 min steps with increasing running speed [14,20]. After each increment to the maximal possible load (running speed max), a capillary blood sample was taken from the earlobe for blood lactate concentration measurement as well as immediately after the termination of the test to obtain the maximal blood lactate concentration. These lactate values were used to calculate the individual anaerobic threshold (IAT) [21]. The running speeds at the described thresholds were transformed into the theoretical time needed individually to reach 1000 m (1000 m time at the IAT). Participants were tested 3 h after breakfast. Blood samples were also drawn prior to performance diagnostics. In addition, the participants were requested to avoid highly intensive physical activity and strength exercise on the previous day to prevent post-training alterations in the pre-test blood samples acquired. 2.5. Training Phase To estimate training compliance and comparability of training volume and exercise in- tensity between the groups, the participants were requested to complete training protocols, documenting weekly training distances and training time over the 12-week intervention phase. The entire training phase was supervised by experienced endurance trainers in both groups. Furthermore, participants were requested to complete a 3-day estimated nutrition protocol before and after the intervention period and were advised to maintain their typical eating and drinking patterns. The evaluation of the food records was performed using the EBISpro nutrition system (Stuttgart, Germany) [22]. 2.6. Statistics Prior to analysis, data were checked for normality distribution. Intragroup compar- isons between baseline and post-intervention as well as before and after the marathon race

intervention period and were advised to maintain their typical eating and drinking patterns. The evaluation of the food records was performed using the EBISpro nutrition system (Stuttgart, Germany) [22]. 2.6. Statistics Prior to analysis, data were checked for normality distribution. Intragroup compar- isons between baseline and post-intervention as well as before and after the marathon race

Nutrients2021,13, 2929 4 of 10 were assessed by the Wilcoxon signed-rank test. Intergroup comparisons were analysed by the Mann–Whitney U test. Spearman's rank correlation analysis was performed to determine the in uence of macronutrient intake on muscle stress reactions. The variables “Diff. Myoglobin ( g/L)” and “Diff. CK (U/L)” were log transformed due to variance in- homogeneity and the analyses (correlation, regression analysis andt-test) were conducted with the transformed variables. SPSS 22.0 (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. All statistical tests were two-sided, and the level of signi cance was set at = 0.05. 3. Results From the 30 endurance athletes (verum group:n= 15, 10 males, 5 females; control group:n= 15, 10 males, 5 females) recruited, 23 completed the 3-month intervention period; no adverse event could be related to the given formula (verum group: 5 dropouts (respiratory tract infection:n= 1, orthopaedic problems:n= 2, personal reasons:n= 2); control group: 2 dropouts (orthopaedic problems:n= 1, personal reason:n= 1)). However, 21 of these 23 athletes were able to perform the marathon race over the complete distance (2 dropouts because of muscle cramps) (Figure).Nutrients 2021, 13, x FOR PEER REVIEW 5 of 11 Figure 1. Study flowchart. Control group: endurance training intervention for 3 months. Verum group: endurance training plus protein-rich supplement for three months. Energy (EI) and macronutrient intakes (expressed as a percentage of EI and relative to BW) in the tested study groups at the beginning and the end of the intervention are presented in Supplementary Table S1. The intervention did not influence the total energy intake in either group. However, the supplement intake resulted in a significant increase in the relative and absolute protein intake in the verum group and yielded a significantly higher protein intake in the intergroup comparisons at the end of the intervention. Given the unchanged total energy intake, the increased protein intake in the verum group was associated with a significant decrease in relative fat intake across the course of interven- tion. Performance and exercise-induced stress variables measured before and after the in- tervention are shown in Table 2. There were no

in the intergroup comparisons at the end of the intervention. Given the unchanged total energy intake, the increased protein intake in the verum group was associated with a significant decrease in relative fat intake across the course of interven- tion. Performance and exercise-induced stress variables measured before and after the in- tervention are shown in Table 2. There were no significant differences in the inter-group comparisons for any of these variables. As a result of the regular training sessions, both running time and running distance accumulated over the 12 weeks of intervention in both groups to a comparable extent (verum group: 3.100 ± 1000 min, 540 ± 230 km; control group: 2.810 ± 570 min, 571 ± 96 km). Aerobic performance showed a slight improvement of approximately 3% in both groups. Table 2. Performance and exercise-induced stress markers measured before (time point a) and after the intervention (time point b). Variable Verum (n = 10) Control ( n = 13) a b a b Max. blood lactate concentration (mmol/L) 8.80 ± 1.20 9.60 ± 1.50 9.20 ± 2.30 10.20 ± 3.60 Running speed max (km/h) 16.4 ± 2.0 16.8 ± 1.9 15.9 ± 1.3 * 16.4 ± 1.5 1000 m-time at the IAT (mm:ss) 04:59 ± 00:42 04:44 ± 00:52 05:12 ±00:31 04:41 ± 00:24 Figure 1. Study owchart. Control group: endurance training intervention for 3 months. Verum group: endurance training plus protein-rich supplement for three months. Basic and anthropometric characteristics of the study groups are presented inTable ; no signi cant differences were found in these variables in the intra- or inter-group com- parisons. Most of the verum group participants achieved a daily protein intake greater than 20% of the total energy intake, based on the 3-day diet diaries indicating intervention compliance regarding the protein supplementation regime.

Nutrients2021,13, 2929 5 of 10 Table 1. Basic and anthropometric characteristics of the study groups (verum, control) before (time point a) and after the 3 months intervention period (time point b). Variable Verum (n= 10) Control ( n= 13) a b a b Sex (m/f) 7/3 9/4 Age (y) 29.0 11.0 28.6 8.7 Body mass (kg) 73.7 6.7 74.5 8.2 71.1 71.5 71.5 8.8 BMI (kg/m 2 ) 23.5 1.2 23.3 1.5 22.4 2.1 22.5 1.9 BFM (%BW) 22.9 9.9 21.8 8.9 22.2 7.7 20.9 7.8 Results are presented as mean SD. There were no statistical differences between both groups. Energy (EI) and macronutrient intakes (expressed as a percentage of EI and relative to BW) in the tested study groups at the beginning and the end of the intervention are presented in Supplementary Table S1. The intervention did not in uence the total energy intake in either group. However, the supplement intake resulted in a signi cant increase in the relative and absolute protein intake in the verum group and yielded a signi cantly higher protein intake in the intergroup comparisons at the end of the intervention. Given the unchanged total energy intake, the increased protein intake in the verum group was associated with a signi cant decrease in relative fat intake across the course of intervention. Performance and exercise-induced stress variables measured before and after the intervention are shown in Table. There were no signi cant differences in the inter-group comparisons for any of these variables. As a result of the regular training sessions, both running time and running distance accumulated over the 12 weeks of intervention in both groups to a comparable extent (verum group: 3.100 1000 min, 540 230 km; control group: 2.810 570 min, 571 96 km). Aerobic performance showed a slight improvement of approximately 3% in both groups. Table 2.Performance and exercise-induced stress markers measured before (time point a) and after the intervention (time point b). Variable Verum (n= 10) Control ( n= 13) a b a b Max. blood lactate concentration (mmol/L) 8.80 1.20 9.60 1.50 9.20 2.30 10.20 3.60 Running speed max (km/h)

km). Aerobic performance showed a slight improvement of approximately 3% in both groups. Table 2.Performance and exercise-induced stress markers measured before (time point a) and after the intervention (time point b). Variable Verum (n= 10) Control ( n= 13) a b a b Max. blood lactate concentration (mmol/L) 8.80 1.20 9.60 1.50 9.20 2.30 10.20 3.60 Running speed max (km/h) 16.4 2.0 16.8 1.9 15.9 1.3 * 16.4 1.5 1000 m-time at the IAT (mm:ss) 04:59 00:42 04:44 00:52 05:12 00:31 04:41 00:24 VO 2max(mL/min/kg) 51.8 6.3 * 53.3 5.9 50.4 4.1 * 51.9 4.9 CK (U/L) 140 120 190 150 170 97 165 95 Myoglobin ( g/L) 35.0 50.0 30.0 15.0 19.0 7.1 18.2 8.0 Leukocytes (1000/ L) 5.3 0.9 5.0 0.3 5.4 1.3 5.2 1.1 IL-6 (pg/mL) 2.0 0.0 2.1 0.2 2.0 0.0 2.1 0.2 Cortisol (ng/mL) 167.00 31.00206.00 58.00 161.00 36.00 * 195.00 45.00 Results are presented as mean SD. *p< 0.05; CK, creatine kinase. Acute exercise-induced stress markers before and 1 h after the marathon race are shown in Table. Twenty-one participants completed the marathon race and were included in the analysis. There was no difference in the marathon running time in the tested groups (verum: 3:54 h 32 min; control: 3:59 h 26 min). Both groups showed signi cant increases in all exercise-induced stress markers. There were trends toward higher calculated increments for all stress markers in the control group. A signi cant inter-group difference was found for serum myoglobin.

Nutrients2021,13, 2929 6 of 10 Table 3.Exercise-induced stress values of the 21 participants who completed both the intervention and the marathon race before (time point c) and after the race (time point d). Variable Verum (n= 9) Control ( n= 12) c d inc. F c d inc. F CK (U/L) 122 41 ** 530 220 4 180 110 ** 1400 1300 8 Myoglobin ( g/L) 26.0 15.0 ** 770 540 † 29 † 32.0 35.0 ** 2200 1800 68 Leukocytes (1.000/ L) 5.3 0.5 ** 15.8 2.5 3.0 5.4 1.1 ** 18.0 4.2 3.3 IL-6 (pg/mL) 2.0 0.0 ** 37.0 30.0 18 2.0 0.1 ** 43.0 15.0 21.3 Cortisol (ng/mL) 184 48.0 ** 302 70.0 1.6 194 48.0 ** 335 95.0 1.7 Results are presented as mean SD. **p< 0.01 (intra-group comparisons between time points).†p< 0.05 (inter-group comparison at time point and for rise factor). Group-speci c increment factor of the measured variables (inc. F.) was calculated as d/c. CK, creatine kinase. Regression analyses among the increases in the measured stress variables and calcu- lated stress score showed that there was a negative association between the individual protein intake (expressed as a proportion of daily protein intake in the post-intervention phase) and the increase in stress markers (signi cant difference in IL-6, cortisol, and stress score; trend for difference in CK, myoglobin, and leukocytes) after the marathon race (Table). The regression curves showed that with a daily protein intake (PI) of less than 20% of the total energy intake, the increase in stress markers was visibly higher. This was statistically con rmed in a group comparison for the participants of the marathon in relation to their protein intake (PI) (n= 12: PI < 20% vs.n= 9: PI = >20%) (Table). The graphical representation based on the stress score (Figure) also showed that an increased protein intake of more than 20% was achieved primarily by participants in the verum group. In contrast to that of the protein intake, the regression analysis did not provide any evidence for a relation between fat and carbohydrate intake and the exercise-induced stress reaction. Table

This study evaluates the effects of protein supplementation on exercise-induced stress in marathon athletes.