The Impact of a Dairy Milk Recovery Beverage on Bacterially Stimulated Neutrophil Function and Gastrointestinal Tolerance in Response to Hypohydration Inducing Exercise Stress

in International Journal of Sport Nutrition and Exercise Metabolism
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  • 1 Monash University
  • 2 Deakin University
  • 3 Writtle University College

The study aimed to determine the impact of a dairy milk recovery beverage immediately after endurance exercise on leukocyte trafficking, neutrophil function, and gastrointestinal tolerance markers during recovery. Male runners (N = 11) completed two feeding trials in randomized order, after 2 hr of running at 70% V˙O2max, fluid restricted, in temperate conditions (25 °C, 43% relative humidity). Immediately postexercise, the participants received a chocolate-flavored dairy milk beverage equating to 1.2 g/kg body mass carbohydrate and 0.4 g/kg body mass protein in one trial, and water volume equivalent in another trial. Venous blood and breath samples were collected preexercise, postexercise, and during recovery to determine the leukocyte counts, plasma intestinal fatty acid binding protein, and cortisol concentrations, as well as breath H2. In addition, 1,000 µl of whole blood was incubated with 1 μg/ml Escherichia coli lipopolysaccharide for 1 hr at 37 °C to determine the stimulated plasma elastase concentration. Gastrointestinal symptoms and feeding tolerance markers were measured preexercise, every 15 min during exercise, and hourly postexercise for 3 hr. The postexercise leukocyte (mean [95% confidence interval]: 12.7 [11.6, 14.0] × 109/L [main effect of time, MEOT]; p < .001) and neutrophil (10.2 [9.1, 11.5] × 109/L; p < .001) counts, as well as the plasma intestinal fatty acid binding protein (470 pg/ml; +120%; p = .012) and cortisol (236 nMol/L; +71%; p = .006) concentrations, were similar throughout recovery for both trials. No significant difference in breath H2 and gastrointestinal symptoms was observed between trials. The total (Trial × Time, p = .025) and per cell (Trial × Time, p = .001) bacterially stimulated neutrophil elastase release was greater for the chocolate-flavored dairy milk recovery beverage (+360% and +28%, respectively) in recovery, compared with the water trial (+85% and −38%, respectively). Chocolate-flavored dairy milk recovery beverage consumption immediately after exercise prevents the decrease in neutrophil function during the recovery period, and it does not account for substantial malabsorption or gastrointestinal symptoms over a water volume equivalent.

The magnitude of the immune response after exercise plays a key role in monitoring, identifying, and clearing pathogenic agents (e.g., environmental pathogens and/or exercise-associated luminal originated bacterial endotoxin translocation) and tissue debris (e.g., soft tissue damage), and in the restabilization of gastrointestinal-associated lymphoid tissue structure and function (Costa et al., 2017b; Peake et al., 2017). These protective and adaptation factors are achieved through patency and stability of the gastrointestinal tract, allowing for nutrient bioavailability, acute innate responses (e.g., neutrophil activity), memorized strategic adaptive responses (e.g., lymphocyte activity), and immune cell communication pathways (e.g., cytokine responses; Walsh et al., 2011). It is well established that endurance exercise has the ability to disturb and/or depress certain structural and functional aspects of immunity, potentially leaving the individual open to opportunistic pathogenic agents and suboptimal recovery processes (Russo et al., 2019; Walsh, 2018). Furthermore, neutrophil chemotaxis, phagocytic ability, pathogenic termination, and/or tissue debris demolition functions (e.g., elastase degranulation and oxidative burst) may play a role in initiating tissue repair (Peake, 2002; Peake et al., 2017; Walsh et al., 2011), which equate to exercise recovery process similarities. However, certain aspects of neutrophil function are consistently shown to be depressed for several hours after endurance exercise (Robson et al., 1999). For example, 2 hr of running at ≥70% V˙O2max consistently results in a >20% depressed in vitro bacterially stimulated (Escherichia coli lipopolysaccharide) neutrophil degranulation (Costa et al., 2019a; Costa et al., 2009; Costa et al., 2011). The precise clinical significance of reduced neutrophil function after endurance exercise is still under debate, considering illness and infection risk is dependent on various factors, including, but not limited to, individuals’ magnitude of resistance and resilience (i.e., individual tolerance level), and the presence and magnitude of internal and/or external originated pathogen exposure (Walsh, 2018; Walsh et al., 2011). Nevertheless, the observed reduction in neutrophil function after endurance exercise is likely associated with the desensitization of mature neutrophils, rather than the release of immature cells from bone marrow storage (Costa et al., 2019a).

Some limited evidence has suggested that carbohydrate (CHO) intake during exercise may prevent the exercise-associated depression in in vitro neutrophil function (Bishop et al., 2002). From a practical and translational perspective, CHO intake during exercise is not always necessary, recommended, and/or tolerated (Burke et al., 2019; Costa et al., 2017a; Costa et al., 2019c). Previous CHO dosages used (e.g., ∼60 g CHO/hr) can be above the total CHO oxidation capacity of individuals, leading to performance-debilitating gastrointestinal symptoms (GIS; Costa et al., 2017a). With this in mind, it has previously been shown that consuming a CHO (1.2 g CHO/kg body mass [BM]) recovery beverage, with or without protein (PRO; 0.4 g PRO/kg BM), from supplement-based ingredients (i.e., maltodextrin and soya PRO) immediately after 2 hr of running at 75% V˙O2max in temperate ambient conditions is able to prevent the postexercise reduction in neutrophil function (Costa et al., 2009; Costa et al., 2011), despite no apparent differences found in blood leukocyte trafficking between feeding interventions (i.e., immediate vs. delayed vs. water control). It was suggested that such an outcome was due to the priming effects of insulin on circulating neutrophils, by the activation of insulin receptors on the neutrophil cell membrane (Walrand et al., 2004).

Recently, there has been much interest in the use of whole foods within sports nutrition application. Considering that the nutritional composition of dairy milk meets the criteria for the general recovery nutrition guidelines and recommendation (Thomas et al., 2016), it is not surprising that the consumption of dairy milk after exercise supports muscle glycogen resynthesis, muscle PRO synthesis, and rehydration processes, which have subsequently translated into enhanced exercise performance outcomes (Russo et al., 2019). However, the role of dairy milk in preventing or ameliorating exercise-induced immunodepression has not been fully explored (Russo et al., 2019). Moreover, considering the essential role of the gastrointestinal tract in the transit, digestion, and absorption of recovery nutrients (i.e., magnitude of nutrient bioavailability), to date, recovery nutrition research has fully neglected to assess gastrointestinal integrity (e.g., epithelial injury, luminal bacteria endotoxin translocation) and functional (e.g., degree of CHO malabsorption) indices (Russo et al., 2019). It is now well established that exercise stress results in intestinal epithelial cell (e.g., enterocyte) injury, evidenced by substantial preexercise to postexercise increases in plasma intestinal fatty acid binding protein (I-FABP) concentration (Costa et al., 2019b; Costa et al., 2017b). In addition, exercise stress has the potential to reduce gastrointestinal transit and enterocyte cell activity through mesenteric and submucosal plexus deactivation linked with the natural stress response of exercise (i.e., increased sympathetic drive; Costa et al., 2019b; Costa et al., 2017b). These two secondary outcomes of exercise-induced gastrointestinal syndrome have the capability of perturbing nutrient trafficking along the gastrointestinal tract and lumen nutrient absorption, subsequently resulting in nutrient malabsorption (Costa et al., 2017a; Lang et al., 2006; van Wijck et al., 2013), previously identified through the H2 and CH4 content of the breath sample for CHO (Bate et al., 2010) and in vivo L-[1-13C] phenylalanine-labeled PRO ingestion with continuous intravenous L-[ring-2H5] phenylalanine infusion technique for PRO (van Wijck et al., 2013). These gastrointestinal patency factors have implications for feeding tolerance, systemic nutrient availability, and subsequent GIS, which are all implicated in the nutritional status for optimizing recovery (Costa et al., 2017a; Costa et al., 2016). Despite dairy milk appearing to be a good exercise recovery option, from a practical perspective, a substantial amount of anecdotal evidence from athletes per se and practitioners suggests dairy milk after exercise is poorly tolerated and leads to severe GIS. As such, dairy milk is generally avoided during exercise recovery by many athletes who do not present a history of lactose and/or milk PRO intolerance.

With this in mind, the current study aimed to (a) determine the impact of a dairy milk beverage immediately after endurance exercise on blood leukocyte trafficking and in vitro bacterially stimulated neutrophil degranulation and (b) determine gastrointestinal tolerance to the dairy milk beverage during the recovery period. It was hypothesized that dairy milk consumption immediately after strenuous endurance exercise would not alter postexercise blood leukocyte trafficking, but would prevent exercise-associated reductions in neutrophil function, compared with the provision of equivalent water volume. In addition, dairy milk consumption immediately after strenuous endurance exercise would lead to greater gastrointestinal intolerance compared with water.

Methods

Participants

A total of 11 non-heat-acclimatized competitive male endurance runners (mean ± SD: age 34 ± 11 years, nude BM 77.2 ± 4.4 kg, height 1.79 ± 0.04 m, percentage of body fat mass 11.0 ± 2.7%, and V˙O2max 59 ± 7 ml·kg BM−1·min−1) volunteered to participate in the study. All participants gave written informed consent. The study protocol received approval from the local ethics committee (CF14/1899-2014000934). The participants were excluded if they confirmed having any gastrointestinal infections, diseases, and/or disorders; consumed potential modifiers of gastrointestinal integrity; had been adhering to gastrointestinal-focused dietary regimes within the previous 3 months; or had consumed nonsteroidal anti-inflammatory medications and/or stool-altering medications within 1 month before the experimental protocol.

Preliminary Measurements

One week before the first experimental trial, height (stadiometer; Holtain Ltd., Crymych, United Kingdom) and nude BM (Seca 515 MBCA; seca GmbH & Co. KG, Hamburg, Germany) were measured. In addition, body composition was determined with an eight-point multifrequency bioelectrical impedance analyzer (Seca 515 MBCA; seca GmbH & Co. KG). The maximal oxygen uptake (V˙O2max; Vmax Encore Metabolic Cart; CareFusion Corp, San Diego, CA) was estimated by means of a continuous incremental exercise test to volitional exhaustion on a motorized treadmill (Forma Run 500; Technogym, Seattle, WA), as previously reported (Costa et al., 2009). To determine running speed for the exercise trials, the speed at 70% V˙O2max and 1% gradient was determined and verified from the V˙O2–work-rate relationship (10.9 ± 1.5 km/hr).

Experimental Procedure

The participants were free to consume their normal diet during their time participating in the study. Dietary intake was recorded for 3 days before each experimental trial through a dietary log. Energy and nutritional intake was assessed and analyzed in accordance with the previously described procedures (Costa et al., 2014; Costa et al., 2013). The participants were asked to refrain from consuming alcohol and caffeinated beverages during the monitoring period and to refrain from strenuous exercise during the 48-hr period before each experimental trial. The participants reported to the laboratory at 0800 hr after consuming the standardized mixed CHO breakfast (300 ml water, 3.1 MJ, 29 g PRO, 15 g fat, and 120 g CHO including starch 50 g, lactose 15 g, fructose 10 g, dextrose 10 g, sucrose 16 g, maltodextrin 10 g, dietary fiber 6 g, and other fermentable oligo-, di-, mono-saccharides, and polyols [FODMAPs] 3 g; modified PowerPackets©, BASE Essentials©, Monash University, Notting Hill, VIC, Australia), with an additional 400 ml of water (consumed at 0700 hr). Before commencing the exercise protocol, the participants were asked to void before the nude BM measurement, provide a breath sample into a 250-ml breath collection bag (Wagner Analysen-Technik GmbH, Bremen, Germany), and complete a validated and reliability-checked exercise-specific modified Visual Analogue Scale GIS assessment tool in real time (Gaskell et al., 2019a). The participants were educated and advised to complete the Visual Analogue Scale as follows: 1–4 indicative of mild GIS (i.e., sensation of GIS, but not substantial enough to interfere with exercise workload) and increasing in magnitude, 5–9 indicative of severe GIS (i.e., GIS substantial enough to interfere with exercise workload), and 10 indicative of extremely severe GIS warranting exercise cessation. If no specific GIS was reported, this was indicative of 0, and subsequently, no rating was warranted. GIS were specified and categorized in accordance with Rome III consensus criteria (Drossman, 2006). Blood was then collected by venipuncture from an antecubital vein (6-ml lithium heparin and 4-ml K3EDTA vacutainers).

The participants completed two experimental trials separated by 1 week, consisting of a 2-hr (initiated at 0900 hr) running exercise on a motorized treadmill (Forma Run 500; Technogym) at the previously determined speed that elicited 70% V˙O2max in 25.2 ± 1.8 °C and 43 ± 9% relative humidity (Figure 1). During running, the participants were totally water restricted to induce a substantial magnitude of hypohydration. The heart rate (Polar Electro, Kempele, Finland), rating of perceived exertion, and GIS were measured every 15 min during exercise in real time. Breath samples were collected hourly during exercise. Immediately postexercise, blood samples were collected, and nude BM was recorded. In a randomized order, the participants were immediately provided with a chocolate-flavored dairy milk recovery beverage (CM) to the value of 1.2 g CHO/kg BM and 0.4 g PRO/kg BM in a 10% CHO wt/vol equivalent (926 ± 53 ml) in one trial, and water (W) volume equivalent in another trial to act as a control and not as a comparator. The pretrial initial assessment nude BM values was used to calculate the CM volume and also the W volume equivalency. The participants were required to complete drinking these beverages within 10 min, as previously conducted (Costa et al., 2009; Costa et al., 2012; Costa et al., 2011). An additional water equivalent to reach 150% of exercise-induced nude BM loss (2,689 ± 633 ml) was provided, and consumption was equally distributed over the 3-hr recovery period in 1-hr blocks. The participants remained seated during the recovery period measured. GIS were measured hourly in real time, and breath samples were collected every 30 min during recovery. Additional blood samples were collected 2-  and 3-hr postexercise. A standard meal and beverage were provided 2 hr into recovery (1.9 MJ, 10 g PRO, 6 g fat, 90 g CHO, and 396 ml water).

Figure 1
Figure 1

—Schematic illustration of the experimental design. BM = body mass; CM = chocolate-flavored dairy milk recovery beverage; GIS = gastrointestinal symptoms; HR = heart rate; Pv = plasma volume; RH = relative humidity; RPE = rating of perceived exertion; Tamb = ambient temperature; W = water.

Citation: International Journal of Sport Nutrition and Exercise Metabolism 30, 4; 10.1123/ijsnem.2019-0349

Sample Analysis

Breath samples (20 ml) were analyzed in duplicate (coefficient of variation [CV]: 4.0%) for H2 content using a gas-sensitive analyzer (BreathTracker Digital MicroLyzer; QuinTron Inc., Milwaukee, WI). K3EDTA whole blood was used to quantify the total and differential leukocyte counts by Coulter Counter (Cabrini Pathology, Malvern, VIC, Australia). Whole blood hemoglobin was determined by a HemoCue system (Hb201; HemoCue AB, Ängelholm, Sweden) in duplicate (CV: 4.3%), and hematocrit was determined by the capillary method, with a microhematocrit reader in triplicate (CV: 1.4%; Thermo Fisher Scientific, Thermo Fisher Scientific Australia, Scoresby, Victoria, Australia), both from heparin whole blood samples. The hemoglobin and hematocrit values were used to estimate changes in plasma volume relative to the baseline and used to correct plasma variables (Dill & Costill, 1974). Blood glucose concentration was determined preexercise and postexercise, using a handheld glucose monitor from heparin whole blood (Accu-Chek Proforma; Roche Diagnostics, Indianapolis, IN), in duplicate (CV: 1.8%). The remaining heparin whole blood samples were centrifuged at 4,000 rpm for 10 min within 15 min of the sample collection, except for 1 ml, which was used to determine the bacterially stimulated neutrophil elastase release. Heparin plasma was aliquoted and stored frozen at −80 °C until analysis, except for 2 × 50-µl plasma, which was used to determine plasma osmolality, in duplicate (CV: 1.3%), by freeze-point osmometry (Osmomat 030, Gonotec; Berlin, Germany). Whole blood was bacterially challenged by combining 1 ml with 50 μl of 1 mg/ml bacterial stimulant (E. coli lipopolysaccharide; Sigma-Aldrich Ltd., Poole, United Kingdom) within 5 min of collection and was gently vortex mixed. The samples were immediately placed in a water bath (Labline; Thermo Fisher Scientific Australia, Scoresby, Australia) at 37 °C for 1 hr of incubation and further mixed by gentle inversion at 30 min. After 1 hr of incubation, the bacterially challenged samples were centrifuged at 4,000 rpm for 10 min. Supernatant was aliquoted and stored frozen at −80 °C for further analysis.

Plasma concentrations of I-FABP (HK406; Hycult Biotech, Uden, The Netherlands), cortisol (RE52061; IBL International, Hamburg, Germany), elastase (BMS269; Affymetrix EBioscience, Vienna, Austria), and sCD14 (HK320; Hycult Biotech) were determined by ELISA. All biovariables were analyzed as per manufacturer’s instructions on the same day, with standards and controls on each plate, and samples from each participant were assayed on the same plate. The CVs for plasma I-FABP, cortisol, elastase, and sCD14 in duplicate were 3.1%, 3.3%, 3.0%, and 6.4%, respectively.

Statistical Analysis

Statistical power calculations were based on the data from previous exercise models reporting disturbances to immune and gastrointestinal function in response to 2 hr of exertional and exertional heat stress (Costa et al 2019a; Costa et al., 2009; Costa et al., 2011; Snipe et al., 2018a, 2018b). Using a standard alpha (.05) and beta value (0.95), a sample size of n = 8, using a randomized cross-over design, is estimated to provide adequate statistical precision to detect variable differences.

Only participants with full datasets within each specific variable were used in the data analysis, as notified in the table and figure legends (i.e., n = 11 for the physiological variable and n = 8 for blood-based variables). The data in the text and tables are presented as either mean ± SD or mean and 95% confidence interval, where indicated. The GIS incidence is presented as a descriptive percentage, whereas GIS severity and feeding tolerance markers are presented as an accumulative score and individual participant range, as previously reported (Gaskell et al., 2019b; Miall et al., 2018; Snipe & Costa, 2018). The data in the figures are presented as mean ± SEM and/or mean + individual responses, as indicated. The variables with singular data points were examined using paired sample t tests or nonparametric Wilcoxon signed-rank test, when appropriate. The variables with multiple data points were examined using a one- or two-way repeated-measures analysis of variance, where appropriate. Assumptions of homogeneity and sphericity were checked, and when appropriate, adjustments to the degrees of freedom were made using the Greenhouse–Geisser correction method. Significant main effects were analyzed using a post hoc Tukey’s honest significance test. Spearman’s rank correlation coefficient was used in the correlation analysis, where indicated. Statistics were analyzed using SPSS statistical software (version 25.0; SPSS Inc., Chicago, IL), with significance accepted at p ≤ .05.

Results

Dietary Intake and Nude BM

There was no significant difference between CM and W for dietary variables. Energy and macronutrient intakes during the monitoring period were (mean [95% confidence interval]) 12.5 [11.2, 13.9] MJ/day (p = .499), 325 [265, 385] g/day CHO (p = .470), 155 [139, 170] g/day PRO (p = .315), 119 [109, 128] g/day fat (p = .285), and 3.3 [2.9, 3.7] L/day water (p = .485). Preexercise (p = .435) and postexercise nude BM was not significantly (p = .502) different between CM (77.3 [74.4, 80.3] kg and 74.9 [72.0, 77.8] kg, respectively) and W (77.1 [74.2, 80.0] kg and 74.7 [71.9, 77.5] kg, respectively). Preexercise nude BM on CM and W did not significantly differ from the pretrial initial assessment (p = .992).

Hydration Status and Physiological Strain

Exercise-induced BM loss was no different between CM and W (Table 1). Plasma osmolality (MEOT; p < .001) increased preexercise to immediately postexercise for CM and W, and returned to similar preexercise baseline values 3 hr into recovery. Similarly, the plasma volume (MEOT; p < .001) decreased preexercise to immediately postexercise for CM and W, returning to the preexercise baseline 2 hr into recovery (Table 1). Despite the rehydration intervention, n = 3 for CM and n = 1 for W did not reestablish full baseline hydration values. An MEOT was observed for the heart rate (p < .001) and rating of perceived exertion (p < .001; Table 1), whereby the heart rate and rating of perceived exertion increased as exercise prolonged.

Table 1

Hydration and Physiological Strain Markers Before, During, and in Recovery From 2-hr Running Exercise at 70% V˙O2max in Temperate Ambient Conditions in Chocolate-Flavored Dairy Milk Beverage and the Water Trials (N = 11)

Strain markersChocolate-flavored dairy milk recovery beverageWater
PreexercisePostexercisePreexercisePostexercise
BM (kg)77.3 [74.4, 80.3]74.9 [72.0, 77.8]77.1 [74.2, 80.0]74.7 [71.9, 77.5]
BM loss (%)3.2 [2.9, 3.4]3.1 [2.6, 3.5]
PreexercisePostexercise2-hr postexercise3-hr postexercisePreexercisePostexercise2-hr postexercise3-hr postexercise
Blood glucose (mMol/L)4.9 [4.2, 5.6]5.2 [4.5, 5.8]5.2 [4.7, 5.7]5.3 [5.0, 5.6]4.7 [3.9, 5.5]5.2 [4.7, 5.8]5.2 [4.9, 5.4]5.5 [5.1, 6.0]
Posmol (mOsmol/kg)294 [288, 300]307 [302, 311]305 [301, 308]299 [295, 304]291 [285, 297]306 [299, 313]298 [289, 306]295 [287, 303]
Δ PV (%)0.0−6.7 [−10.5, −2.9]+1.7 [−2.8, 6.2]+2.8 [−0.5, 6.1]0.0−5.8 [−8.3, −3.3]+2.7 [0.6, 4.7]+4.4 [2.8, 6.1]
During: 30 min60 min90 min120 minDuring: 30 min60 min90 min120 min
HR (bpm)142 [133, 152]145 [136, 154]149 [141, 158]158 [148, 168]144 [133, 155]146 [136, 156]151 [141, 161]159 [149, 169]
RPE (6–20)11 [11, 12]12 [12, 13]14 [13, 15]15 [13, 16]12 [11, 12]13 [12, 14]14 [13, 15]15 [13, 17]

Note. The values are presented in mean [95% confidence intervals]. BM = body mass; RPE = rating of perceived exertion; Pv = plasma volume; Posmol = plasma osmolality; HR = heart rate.

Plasma Cortisol and Blood Glucose Concentration

No significant main effects or interactions were observed for blood glucose concentration (Table 1). An MEOT (p = .006) was observed for plasma cortisol concentration, whereby levels increased pre-exercise to immediately postexercise for CM and W (Figure 2a).

Figure 2
Figure 2

—Preexercise to immediately postexercise change in plasma cortisol (a), I-FABP (b), and SCD14 (c) concentrations in response to 2-hr running exercise at 70% V˙O2max in temperate ambient conditions in CM and the W trials. I-FABP = intestinal fatty acid binding protein; MEOT = main effect of time; CM = chocolate-flavored dairy milk recovery beverage; W = water. Mean and individual responses (n = 8): MEOT: ††p < .01 and p < .05 versus preexercise (0 min).

Citation: International Journal of Sport Nutrition and Exercise Metabolism 30, 4; 10.1123/ijsnem.2019-0349

Gastrointestinal Integrity and Symptoms

An MEOT (p = .012) was observed for plasma I-FABP concentration, whereby levels increased preexercise to immediately postexercise for CM and W (108%; Figure 2b). No significant main effects or interactions were observed for plasma sCD14 concentration (Figure 2c). An MEOT was observed for breath H2 concentration (p = .004). Breath H2 concentration significantly increased at 3 hr of postexercise for CM and W (Figure 3). No trial difference in area under curve was observed for breath H2 concentration (CM: 1,249 [314, 2,179] ppm/3 hr and W: 1,188 [565, 1,812]ppm/3 hr; p = .878).

Figure 3
Figure 3

—Breath H2 in response to 2-hr running exercise at 70% V˙O2max in temperate ambient conditions in CM and the W trials, including the consumption of a standardized meal containing 120 g mixed CHO 2 hr before exertional stress. (a) Mean ± SEM (CM: ♦ and W: ◊), (b) mean (▪) and individual responses (•) (n = 11): MEOT ††p < .01 versus immediately postexercise (120 min of exercise). CM = chocolate-flavored dairy milk recovery beverage; W = water; CHO = carbohydrate; MEOT = main effect of time.

Citation: International Journal of Sport Nutrition and Exercise Metabolism 30, 4; 10.1123/ijsnem.2019-0349

Running for 2 hr at 70% V˙O2max in temperate ambient conditions on two separate occasions that resulted in mild hypohydration caused a 78% incidence of total GIS (Table 2). CM postexercise resulted in 45% GIS incidence, whereas volume equivalent W resulted in 36% incidence of total GIS. There was no significant difference in GIS severity during exercise and in recovery between CM and W. Significant correlations between breath H2 with total GIS (rs = .422, p = .050) and lower GIS (rs = .465, p = .029) were observed. No difference in feeding tolerance was observed during exercise for the CM and W trials, except for “tolerance to food” that was higher for CM (Table 2). During recovery, CM resulted in significantly lower feeding tolerance, but not “thirst.”

Table 2

Incidence and Severity of GIS and Feeding Tolerance Markers During and in Recovery From 2-hr Running Exercise at 70% V˙O2max in Temperate Ambient Conditions in Chocolate-Flavored Dairy Milk Recovery Beverage and the Water Trials

Chocolate-flavored dairy milk recovery beverageWater
GIS and feeding tolerance markersIncidence during (%)Severity duringIncidence recovery (%)Severity recoveryIncidence during (%)Severity duringIncidence recovery (%)Severity recovery
Gut discomfortNA106 (8–22)NA27 (1–10)NA135 (1–23)NA9 (1–4)ns
Total GISa72222 (8–101)4564 (1–30)82240 (1–69)3611 (1–5)ns
Upper GISb6495 (7–46)3624 (1–12)6451 (1–21)00 (0–0)ns
 Belching5439 (2–12)182 (1–1)4524 (1–11)00 (0–0)ns
 Heartburn1819 (6–13)91 (1–1)185 (1–4)00 (0–0)ns
 Bloating91 (1–1)1810 (2–5)92 (2–2)00 (0–0)ns
 Stomach pain1822 (5–17)185 (1–4)183 (1–2)00 (0–0)ns
 Urge to regurgitate2714 (1–11)186 (1–5)917 (17–17)00 (0–0)ns
 Regurgitation00 (0–0)00 (0–0)00 (0–0)00 (0–0)ns
Lower GISb4529 (3–11)2737 (2–30)5456 (1–20)93 (3–3)ns
 Flatulence3620 (1–8)1812 (2–10)1825 (8–17)93 (3–3)ns
 Urge to defecate93 (3–3)1813 (3–10)91 (1–1)00 (0–0)ns
 Lower abdominal pain96 (3–3)1812 (2–10)3630 (3–12)00 (0–0)ns
 Abnormal defecationc00 (0–0)00 (0–0)00 (0–0)00 (0–0)ns
Others
 Nausea3624 (1–21)00 (0–0)3644 (8–23)185 (1–4)ns
 Dizziness4546 (3–17)91 (1–1)4568 (2–22)273 (1–1)ns
 Abdominal stitchd1828 (11–17)182 (2–2)2721 (6–8)00 (0–0)ns
Feeding tolerance
 Interest in foodNA138 (2–27)NA123 (7–20)NA103 (4–22)NA208 (15–28)**
 Interest in drinkNA472 (34–62)NA106 (5–20)NA448 (29–70)NA160 (9–30)***
 Tolerance to foodNA314 (6–73)NA179 (8–28)NA237 (1–76)NA252 (15–30)*,**
 Tolerance to drinkNA609 (43–80)NA161 (6–30)NA523 (23–86)NA205 (12–30)***
 AppetiteNA148 (4–31)NA135 (5–22)NA143 (4–47)NA214 (13–30)**
 ThirstNA506 (36–66)NA117 (4–21)NA490 (30–70)NA128 (7–22)ns

Note. Overall participant summative accumulation of measured time periods and individual range of those participants reporting symptom (n = 11). NA = not applicable; GIS = gastrointestinal symptoms.

Trial difference during exercise: *p < .05; during recovery: **p < .01 and ***p < .05; and NS indicates no significance.

aSummative accumulation of upper, lower, and other GIS. bSummative accumulation of upper or lower GIS. cAbnormal defecation including loose watery stools, diarrhea, and blood in stools. dAcute transient abdominal pain.

Total and Differential Leukocyte Counts and Neutrophil Functional Response

An MEOT was observed for total leukocyte (p < .001) and neutrophil (p < .001) counts. The total leukocyte and neutrophil counts increased preexercise (6.0 [5.4, 6.6] × 109/L and 3.5 [3.1, 3.9] × 109/L, respectively) to immediately postexercise (10.5 [9.5, 11.6] × 109/L and 7.0 [6.0, 8.1] × 109/L, respectively) and remained elevated throughout the recovery period (12.7 [11.6, 14.0] × 109/L and 10.2 [9.1, 11.5] × 109/L, respectively) in both trials. An MEOT (p = .003) was also observed for the neutrophil to lymphocyte ratio, whereby the ratio increased preexercise (2.2 [1.9, 2.5]) to immediately postexercise (2.9 [2.3, 3.6]), peaking at 2 hr into recovery (8.0 [6.5, 11.1]) in both trials. A Trial × Time interaction was observed for unstimulated (p = .015) and stimulated (p = .025) neutrophil elastase release, whereby the concentrations increased preexercise to immediately postexercise for CM and W, remaining elevated throughout the recovery period in both trials, but were higher for CM (Figure 4a and 4b). A Trial × Time interaction (p = .001) was observed for bacterially stimulated elastase release per neutrophil during the recovery period. CM resulted in a significant increase in neutrophil functional responses (+27%), whereas a −38% depression was observed at 3 hr of postexercise for W (Figure 4c).

Figure 4
Figure 4

—Unstimulated (a), stimulated (b), and per cell (c) bacterially stimulated neutrophil elastase release in response to 2-hr running exercise at 70% V˙O2max in temperate ambient conditions in CM (•) and the W (▪) trials. (i) Mean ± SEM (CM: ♦ and W: ◊), (ii) mean (▪) and individual responses (•) (n = 8): **p < .01 and *p < .05 versus preexercise (0 min), and aap < .01 and ap < .05 versus W. CM = chocolate-flavored dairy milk recovery beverage; W = water.

Citation: International Journal of Sport Nutrition and Exercise Metabolism 30, 4; 10.1123/ijsnem.2019-0349

Discussion

The current study aimed to determine the impact of a dairy milk beverage immediately after endurance exercise on leukocyte trafficking and in vitro bacterially stimulated neutrophil degranulation, using water volume equivalency as a control. In addition, the study aimed to determine gastrointestinal tolerance to the dairy milk beverage during the recovery period. In accordance with our hypothesis, consuming a dairy milk beverage (1.2 g CHO/kg BM + 0.4 g PRO/kg BM) immediately after a 2-hr running exercise at 70% V˙O2max in temperate ambient conditions and resulting in hypohydration (i.e., 3.2% BM loss, plasma osmolality 307 mOsmol/kg, and Δplasma volume −6.3%) did not alter blood leukocyte trafficking compared with water volume equivalent. However, consuming a dairy milk beverage immediately after the exercise bout did prevent the exercise-associated depression in neutrophil function, as assessed by in vitro bacterially stimulated neutrophil degranulation (i.e., elastase release). In contrast to our hypothesis, dairy milk beverage consumed at the initial phase of recovery resulted in lower feeding tolerance markers during recovery, but did not result in substantial CHO malabsorption and GIS compared with the water volume equivalent.

The authors acknowledge that the current study did not include, and was not focused on including, an isocaloric comparator trial (e.g., CHO + PRO supplement recovery beverage), as previously investigated (Costa et al., 2009; Costa et al., 2012; Costa et al., 2011). Thus, direct comparison with previous recovery nutrition isocaloric supplementation intervention research warrants caution. In addition, the consumption of a recovery beverage containing CHO, PRO, and water is recommended soon (e.g., <1 hr) after hypohydration-inducing endurance exercise to aid optimal recovery (Russo et al., 2019; Thomas et al., 2016). Consistent with previous experimental models, the dairy milk beverage intake volume applied in the current study (i.e., 926 ml within 10 min) may not represent the real-life application of recovery nutrition practice in all athlete groups, but it is an identified practice, especially in endurance and ultraendurance sports (Costa et al., 2019c; Costa et al 2016; Thomas et al., 2016). Such a nutrient-dense volume may cause GIS and feeding intolerance along the recovery period in some individuals. Therefore, recovery GIS outcomes (Table 2) may be overinflated, due to n = 1 high responder, compared with a feeding delivery spread evenly over a long time period (e.g., 309 ml every 10 min for 30 min). The authors also acknowledge the limitation of the in vitro model used to establish changes in neutrophil function respective to practical translation application to in vivo models. Recent in vivo immune responses through hypersensitivity challenge with diphenylcyclopropenone antigen have been explored in exercise models, with and without CHO intervention (Davison et al., 2016; Diment et al., 2015). This method, however, is not specific to acute and transient immune functional responses targeting lumen-originated bacterial endotoxin translocation into the systemic circulation and pathophysiology of exercise-induced gastrointestinal syndrome, in which neutrophil functional responses and adjunct cytokine interactions play a role in managing such potential clinically significant outcomes (e.g., exercise-associated systemic inflammatory response syndrome [SIRS] and septic shock; Costa et al., 2019b; Gill et al., 2015b; Peake, 2002; Peake et al., 2015). The development of accurate and reliable in vivo neutrophil functional biomarkers would facilitate the translational application of the current research finding, with interpretation/s made in conjunction with gastrointestinal injury, systemic bacterial endotoxin, and inflammatory markers.

It is generally recognized that exercise creates an immunodepressive scenario in the recovery period, creating an open window for opportunistic pathogenic microorganisms (Peake et al., 2017). For example, a 23–31% reduction in stimulated neutrophil function has previously been observed in response to 2-hr running at 75% V˙O2max in 20 °C (Costa et al., 2009; Costa et al., 2011). It is, however, less reported that luminal-originated bacteria, bacterial endotoxins (e.g., lipopolysaccharide), and other immunomodulating bacterial components (e.g., peptidoglycan, lipid A, and outer membrane vesicles) have the potential to translocate into systemic circulation, generally peaking immediately or 1-hr postexercise, if exercise stress is substantial enough, and contribute to the overall pathogenic load (Costa et al., 2017b; Gill et al., 2015a, 2015b; Snipe et al., 2018a, 2018b). Adequate recovery nutrition counteracts some of the exercise-associated immune disturbances (Costa et al., 2009; Costa et al., 2012; Costa et al., 2011), including attenuating reductions in neutrophil function to a bacterial challenge (i.e., E. coli lipopolysaccharide), which appears to be an important functional response for clearance of exercise-associate systemic endotoxemia, but the functional translation into in vivo human models requires substantiation (Davies et al., 2019; Peake, 2002; Peake et al., 2017). The current study showed that the provision of a dairy milk beverage, with an equivalent macronutrient profile to previous research, prevented the drop in stimulated neutrophil function, as seen in W (−38%), but also resulted in an increase at 3-hr postexercise (+27%). It is speculated that the dairy milk insulinotropic effect on polymorphonuclear neutrophil cell membranes’ insulin receptors may have resulted in priming neutrophil chemotaxis, phagocytosis, and bactericidal capacity (Walrand et al., 2004; Walrand et al., 2006). In addition, the acute provision of calcium within the diary milk bolus may have enhanced the effectiveness of neutrophil phagocytosis and degranulation processes (e.g., calcium-dependent intracellular targeting and fusion of phagocyte granules, including delivery of elastase to the phagosome; Jaconi et al., 1990; Tapper et al., 2002).

Endurance exercise is associated with intestinal epithelial injury, which may reduce intestinal nutrient transport, gut-associated lymphoid tissue (e.g., antimicrobial PRO and immunoglobulin secretions), and mucosa-associated lymphoid tissue (e.g., goblet cell mucosal secretions) functional responses due to loss of structural integrity and (or) sympathetic-drive-prompting epithelial cell deactivation and/or potential apoptosis (Costa et al., 2019b; Costa et al., 2017b; Grootjans et al., 2016; Holzer et al., 2017). The exertional stress applied resulted in a modest 120% increase in plasma I-FABP concentration (i.e., 470 pg/ml), in comparison with exertional heat stress (e.g., >1,000 pg/ml; Snipe & Costa, 2018; Snipe et al., 2018a; Gaskell et al., 2019b). It is, therefore, not surprising that plasma sCD14 concentration (i.e., indirect marker of luminal endotoxin translocation) was not substantially perturbed. Nevertheless, these modest disturbances to intestinal integrity may have been sufficient to instigate the substantial CHO malabsorption (i.e., >10 ppm breath H2) of the preexercise meal and/or recovery beverage (Bate et al., 2010), observed with CM and W, which may have contributed to the incidence and severity of the GIS reported (Costa et al., 2019a; Costa et al., 2017a; Gaskell et al., 2019b; Miall et al., 2018). On that note, common anecdotal belief states that consuming dairy milk in the postexercise recovery period creates greater GIS than other beverage consumption (e.g., recovery beverage formulation, CHO electrolyte beverage, or water). The incidence of GIS in recovery was similar with CM (45%) and W (36%), with varying symptom types. Despite a higher severity of GIS with CM, no significant difference between CM (total GIS range of responders: 1–30) and W (1–5) was observed, likely due to the large individual variation, predominantly prompted by one participant. Such results indicate the potential for large heterogeneity tolerance to dairy milk beverage postexercise, even in a participant cohort that reports no history of gastrointestinal disease/disorder and no intolerance or GIS to dairy milk consumption at rest. From a translational perspective, individual response will dictate the application of such a recovery beverage in the postexercise period, thus supporting an individualized, tailored recovery nutrition approach.

With respect to CM, a visible increase in peak breath H2 was observed toward the end of the recovery period measures (21 ppm at 2 hr). This was attributed to one participant with breath H2 of 128 ppm, clearly showing malabsorption of the dairy milk recovery beverage (peak individual breath H2 in recovery ranging from 3 to 128 ppm and 2 to 50 ppm for CM and W, respectively). These findings have clear practical implications, suggesting that all historical recovery research conducted may have underestimated the full potential of the tested recovery beverage. These previous studies have failed to identify gastrointestinal patency within their experimental designs, biomarker analysis, and results interpretation; thus, “recovery optimization” has not yet been fully explored (Russo et al., 2019). Moreover, it is still unknown whether the differing energy and macronutrient compositions, wt/vol concentrations, bolus volume, frequency, and timing of bolus would alter gastrointestinal patency and circulating nutrient availability in the recovery period and, subsequently, impact the immune status (e.g., mucosal, innate and adaptive functional responses, systemic endotoxin clearance and inflammatory cytokine responses) during this period of potential heightened illness and (or) infection risk.

Conclusion

Dairy milk beverage consumed immediately after endurance exercise prevented exercise-associated depression in in vitro bacterially stimulated neutrophil function, but did not influence postexercise leukocyte trafficking. The CHO malabsorption and GIS observed with dairy milk consumption in recovery did not substantially differ from water alone. These results suggest a dairy milk beverage consumed as a recovery beverage may support immune competency during a potentially immune-compromised period and is well tolerated from a gastrointestinal perspective.

Acknowledgments

First, the authors would like to thank all the participants who volunteered to take part in this study. The authors’ contributions are as follows: R. Costa was the chief investigator of this research and contributed toward the original research idea and development of the experimental design. All other authors contributed toward various aspects of data collection, sample collection, and analysis. R. Costa and V. Camões-Costa contributed to the analysis of the raw data. All authors contributed to the preparation and review of the article. All authors read and approved the final article. The research study was supported by the BASE Facility, Department of Nutrition Dietetics & Food, Monash University. R. Costa is the intellectual property owner of the BASE Essential© meal range.

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Costa, Russo, and Huschtscha are with the Department of Nutrition, Dietetics and Food, Monash University, Notting Hill, VIC, Australia. Camões-Costa is with the Department General Practice, Monash University, Notting Hill, VIC, Australia. Snipe is with the Centre for Sport Research, School of Exercise and Nutrition Sciences, Deakin University, Burwood, VIC, Australia. Dixon is with the Writtle University College, Lordship Road, Chelmsford, United Kingdom.

Costa (ricardo.costa@monash.edu) is corresponding author.
  • View in gallery

    —Schematic illustration of the experimental design. BM = body mass; CM = chocolate-flavored dairy milk recovery beverage; GIS = gastrointestinal symptoms; HR = heart rate; Pv = plasma volume; RH = relative humidity; RPE = rating of perceived exertion; Tamb = ambient temperature; W = water.

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    —Preexercise to immediately postexercise change in plasma cortisol (a), I-FABP (b), and SCD14 (c) concentrations in response to 2-hr running exercise at 70% V˙O2max in temperate ambient conditions in CM and the W trials. I-FABP = intestinal fatty acid binding protein; MEOT = main effect of time; CM = chocolate-flavored dairy milk recovery beverage; W = water. Mean and individual responses (n = 8): MEOT: ††p < .01 and p < .05 versus preexercise (0 min).

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    —Breath H2 in response to 2-hr running exercise at 70% V˙O2max in temperate ambient conditions in CM and the W trials, including the consumption of a standardized meal containing 120 g mixed CHO 2 hr before exertional stress. (a) Mean ± SEM (CM: ♦ and W: ◊), (b) mean (▪) and individual responses (•) (n = 11): MEOT ††p < .01 versus immediately postexercise (120 min of exercise). CM = chocolate-flavored dairy milk recovery beverage; W = water; CHO = carbohydrate; MEOT = main effect of time.

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    —Unstimulated (a), stimulated (b), and per cell (c) bacterially stimulated neutrophil elastase release in response to 2-hr running exercise at 70% V˙O2max in temperate ambient conditions in CM (•) and the W (▪) trials. (i) Mean ± SEM (CM: ♦ and W: ◊), (ii) mean (▪) and individual responses (•) (n = 8): **p < .01 and *p < .05 versus preexercise (0 min), and aap < .01 and ap < .05 versus W. CM = chocolate-flavored dairy milk recovery beverage; W = water.

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