Naclerio, F., Larumbe-Zabala, E., Ashrafi, N., Seijo, M., Nielsen, B., Allgrove, J., & Earnest, C. P. (2016). Effects of protein–carbohydrate supplementation on immunity and resistance training outcomes: a double-blind, randomized, controlled clinical trial. European Journal of Applied Physiology, 117(2), 267-277. doi:10.1007/s00421-016-3520-x
Note: The funding for this study was provided by MEATPROT and the University of Greenwich
Introduction: Intense exercise can weaken the body’s immune system leading to increased risk for infections and sickness. This immunosuppression has been demonstrated to last up to 72 h post exercise. One method to potentially combat this immunosuppression is to ingest certain nutrients post-workout. In particular, a combination of carbohydrates and high quality protein ingested during the day or post-workout is proposed as a means to mitigate immunosuppression. Additionally, protein intake provides amino acids to augment resistance training outcomes. This increased intake of protein results in an improved protein balance and muscle anabolism, thus potentially increasing hypertrophy and strength. Whey and beef protein sources include sulfur containing amino acids, notably cysteine, methionine, and taurine. These amino acid sources are associated with an improved immune status. Therefore, it is conceivable ingesting high quality protein sources could support the immune response in individuals engaging in exhaustive exercise programs. Defensins are antimicrobial peptides (AMP) which contribute to the mucosal host defense system by providing a broad spectrum of antibacterial and fungal activities. Alpha-defensins are known as human neutrophil peptides (HNP1-3) are found in neutrophils. These peptides have been analyzed in multiple disease models. To date, only two studies have investigated exercises effects on HNP1-3, with both demonstrating acute increases in HNP1-3. This post-exercise increase in AMP may be related to the exercise-induced muscle inflammatory response. Airway inflammation and potential damage to epithelial cells may also induce AMP. Interventions aimed at improving humoral immunity without side effects are of relevance to athletes. The purpose of the current study was to investigate the effects of hydrolyzed beef protein or whey protein in combination with carbohydrates verses carbohydrate only on resistance training outcomes and alpha-defensins (HNP1-3). Secondary outcomes will include performance, body composition, muscle thickness, and blood indices of health.
The introduction was done very well. The authors touched on how exercise affects the immune system. In particular, they discussed how alpha-defensins have been analyzed in multiple disease models but have yet to be explored following exercise combined with supplementation. It will be interesting to see the results of this study, as increased risk for respiratory infections is one of the hallmark signs of increased training and potential over-training. If protein and carbohydrates can help reduce this risk then post-workout supplementation may be worth it regardless if there is an impact on LBM and body composition.
Subject Description: Forty- two recreationally active college subjects met the requirements for the study (24 m, 18 f). Inclusion criteria included 18-40 y, regularly training for the past 2 years with at least 1 month of resistance training, free from injury, agreement to avoid supplements, English speaking. Exclusion criteria included history of metabolic disease, medication, use of steroids or creatine, amino acids, whey protein, DHEA, glutamine within the past 8 weeks, use of tobacco or oral contraceptives. Twenty-seven of the 42 subjects completed the study.
The authors did a good job of describing the exclusion and inclusion criteria for the subjects. Not all studies include this information and it is important for the reader as it gives outsiders an idea of the study population. In this case, the reader can see the individuals were active, but may not necessarily have much experience with resistance training with 1 month experience being the criteria. Overall, there was 5 individuals whom ended up lost to follow-up in each group, for a total of 15 people being lost to follow-up. This attrition rate is rather high and can be frustrating for researchers. The age and other demographics are provided later in the study.
Methods: This study was a randomized control trial with three groups, with a double blind between participant design. Subjects were randomly allocated to one of three groups: beef protein (n=14), whey protein (n=14), or carbohydrate only (CHO, n=14). The study was 8 weeks in duration and before and after testing included measurements of performance, body composition, muscle thickness, measures of health, muscle damage, and HNP1-3 salivary immunepeptides. Six familiarization sessions were performed to minimize the learning effect of the training procedures. Subjects were then matched by gender and training background and then randomly allocated. Group characteristics were: beef: age 25.6 ± 5.7 years, height 1.72 ± 0.09 m, body mass 74.2 ± 17.3 kg; whey: age 25.9 ± 5.9 years, height 1.71 ± 0.10 m, body mass 70.2 ± 11.3 kg; CHO: age 24.9 ± 8.1 years, height 1.70 ± 0.09 m, body mass 70.4 ± 15.3 kg. These measurements were not statistically different from one another.
Supplementation included all three groups receiving 20 g sachets of vanilla-flavored powder which was mixed in cold orange juice. The drinks were similar in appearance, texture, and taste and were isoenergetic. Products were taken for 8 weeks at one time per day. On training days the supplement was consumed immediately after training. On non-training days the supplement was ingested in the morning. The beef and whey protein supplements contained ~ 25g of protein and carbohydrates. The carbohydrate supplement contained ~45 g carbohydrates. All three totaled ~ 180 calories. A research nutritionist collected dietary habits and each subject completed a 3 day food diary report. Subjects were asked to maintain their normal diet.
Body mass (BM) and height were recorded on a standard scale. Whole body densitometry was assessed via Bod Pod. Complete body composition was performed twice, if agreement on percentage of body fat was 0.05% then the two tests were averaged. If the test was not within this range a third test was performed and then all three were averaged. Subjects were positioned supine with the leg fully extended and in a relaxed state. The probe was placed 80% of the distance from the greater trochanter to the lateral condyle of the femur. Three images were taken and the average used to determine thickness.
Subjects participated in resistance training three times per week for 8 weeks. Sessions were carried out in the afternoon/evening and subjects completed three circuits including one set of 8 exercises: jump-squat, bench press, parallel back squat, upright row, dumbbell alternate lunges, shoulder press, lateral hurdle jumps, and abdominal crunch. Each exercise was performed for 12 repetitions using the heaviest load possible with the exception of the crunch, which was performed for 20 repetitions. Minimum rest periods were permitted between exercises. Rest between circuits was 2-3 minutes. Total load (kg) summarized for the first six exercises was used to determine performance. Testing occurred during/after the first and last training sessions.
Blood chemistry samples were taken before and after the 8 week periods. These samples were taken in a fasted stated on two occasions, 1 day prior and 1-2 days post intervention. Samples were taken using a finger prick capillary. Measurements were obtained for fasting total cholesterol, HDL, LDL cholesterol triglycerides, and glucose, CK, creatinine, AST/GOT, ALT/GPT, urea and uric acid.
Saliva was collected pre and post initial training session and last training session. Saliva was taken within 5 minutes post training. Each sample was taken between 2 and 7 pm to avoid any changes due to circadian rhythm. Samples were analyzed using ELISA.
The methods section was done well with detail and explanation of the testing procedures. I felt the authors did a good job of explaining the testing procedures as well as choosing multiple measurements and performance variables. Through their testing they are essentially able to decipher whether whey, beef, or carbohydrates is more beneficial for performance. Additionally they are able to decipher which if any have immune benefits and possibly improve (or impair) health indices. One issue I have with the methods is again I feel like the training protocol is not designed to generate hypertrophy or reductions in body fat. It seems like to me it would almost be a maintenance program. The program is not periodized, there are not planned increases, tapers, and altered volume/intensity. There was no baseline strength testing done, therefore it would be hard to prescribe % RM for the individuals to select appropriate weight.
Results: Sample size was calculated using a model of three groups with four repeated measures a 0.05 error probability and a 08. power. Effect sizes were small d=0.2, moderate d=0.5, and large d=0.8).
Fifteen subjects dropped out of the study (9 male, 6 female). Twenty-seven participants, 15 male and 12 female completed the study. The sample was determined to be adequate through power analysis.
Combined resistance training with any of the interventions did not produce statistically significant differences between the three treatment conditions in any of the analyzed variables. Beef showed large effect sizes for body mass (d=1.27), total fat-free mass (d=0.75), vastus medialis thickness (d=1.93), and total kg lifted (d=2.16). CHO demonstrated large effect sizes for total kg lifted (d=1.73) and vastus medialis thickness (d=1.04). Whey demonstrated a large effect size for total kg lifted (whey d=0.97).
In regards to salivary alpha-defensins, significant supplement-time interaction effect for the concentration rates (p=0.031) along with significant effect of time (p=0.053). There were no differences between treatment conditions (p=0.533). No significant interaction (p=0.215), treatment condition (p=0.591), or time effect (p=0.120) was found for secretion rates. Beef demonstrated significant decrease of the baseline concentration and secretion rates of HNP1-3 from values measure prior to the first session (1.90 ± 0.83 μg/mL; 2.95 ± 2.83 μg/min) in comparison to before (0.92 ± 0.63 μg/mL, p = 0.025, d = 1.03; 0.76 ± 0.74 μg/ min, p = 0.049, d = 0.95), and after (0.95 ± 0.60 μg/ mL, p = 0.032, d = 1.00; 0.59 ± 0.52 μg/min, p = 0.027, d = 1.02) performing the last session.
No significant interaction or treatment effect was observed for salivary flow rate (p=0.193, p=0.633). A significant time effect was found (p=0.040). No significant differences were found between groups at any time point.
Discussion: The results of this investigation indicate consuming a post-workout carbohydrate-protein (beef or whey) during an 8 week resistance training intervention promote similar performance and body composition benefits in comparison to carbohydrate. The main finding of this study was the reduction in baseline levels and secretion rates of salivary-defensins HNP-1-3 after the 8 week intervention in the beef group. A non-significant trend was observed in the whey protein group, while no changes were noted in the carbohydrate group. There were no changes in health indices in any of the groups. This is important as both whey and beef groups had significantly increased protein intakes. Despite the increase in protein, the total protein intake was within the 1-2 g/kg-1 recommendation. This is consistent with recent research demonstrating safety of a high protein diet over a short time period (4 months). The post-workout supplementation of protein did not appear to augment resistance training adaptation. All three groups demonstrated similar improvements in body composition, muscle mass, and kg lifted. The beef group appeared to demonstrate a slightly superior body mass gain based on fat-free mass accretion, which was demonstrated by the largest effect size for vastus medialis thickness. This is the first study to compare the effects of a hydrolyzed beef protein powder in comparison to whey protein. Total energy intake was also greatest in the beef group, which demonstrates total caloric intake may be one of the most relevant factors for training adaptation. The results also failed to demonstrate an acute increase of HNP1-3 after exercise at before and after the intervention period. Previous studies demonstrating increases of HNP1-3 examined submaximal cycling, which may explain for the difference in the results. This type of exercise may stress the airways or neutrophilia of the blood, which can increase HNP1-3. Endurance exercise has been demonstrated to activate neutrophils, which may lead to expression in saliva. Another reason for the lack of salivary AMP may be due to the subjects being reasonably well-trained. It is possible the initial reduction in salivary AMP may be an adaptive response to exercise, which may not occur in trained individuals. This response would be a reflection of a reduced immune competence. Increased susceptibility to infection is well-reported in well-trained athletes. It is important to note, all participants but one in the beef depicted a consistent decrease in HNP1-3 from pre to post intervention. In whey, three participants and in CHO, two participants demonstrated a decrease in HNP1-3. The post-intervention decrease of HNP1-3 in the two protein groups may be due to the reduced carbohydrate intake. This would support carbohydrate intake post-exercise as an effective method to countermeasure exercise-induced acute immune dysfunction. Although this observation should be interpreted with caution, as the majority of research in this area includes only acute studies. The present study demonstrated a reduction in HNP1-3 over an 8 week intervention, which is considered a medium to long term adaption similar to other salivary markers in well-trained athletes. The lower levels in the beef group could be interpreted as a positive response, meaning individuals stress levels were reduced. Decreased HNP1-3 has been implicated as a reduced risk factor against virus and infection. In summary, the current study suggests the ingestion of a combined protein and carbohydrate supplement post-workout supports some adaptions induced by resistance training. Additionally, the combination of protein and carbohydrate resulted in an early decrease of salivary alphadefensins (HNP1-3).
Conclusion: In conclusion, a combination of protein and carbohydrates post-workout resulted in positive adaptation and a reduced HNP1-3 level. Future research should look to confirm this theory and explore its connection with infectious diseases in athletes.
My Thoughts: This was a well done study, with the authors exploring multiple endpoints. I was not a fan of the training program, and thought it could have been planned better. The authors did a good job of linking the beef protein group with overall higher calorie intake. It will be interesting to see future research compare hydrolyzed beef protein to whey. Regardless of the calorie intake, it was interesting to see the beef protein group have superior muscle thickness and gains in fat free mass. The whey group demonstrated a trend to lose more body fat. It was surprising whey did not appear to influence HNP1-3 levels as much as beef protein, as whey is often touted for its immune benefits. It will also be noteworthy for future research to determine if nutrient timing had anything to do with these results.
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