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ARTICLE Effects of winter military training on energy balance, whole-body protein balance, muscle damage, soreness, and physical performance Lee M. Margolis, Nancy E. Murphy, Svein Martini, Marissa G. Spitz, Ingjerd Thrane, Susan M. McGraw, Janet-Martha Blatny, John W. Castellani, Jennifer C. Rood, Andrew J. Young, Scott J. Montain, Yngvar Gundersen, and Stefan M. Pasiakos
Abstract: Physiological consequences of winter military operations are not well described. This study examined Norwegian soldiers (n = 21 males) participating in a physically demanding winter training program to evaluate whether short-term military training alters energy and whole-body protein balance, muscle damage, soreness, and performance. Energy expenditure (D218O) and intake were measured daily, and postabsorptive whole-body protein turnover ([15N]-glycine), muscle damage, soreness, and performance (vertical jump) were assessed at baseline, following a 4-day, military task training phase (MTT) and after a 3-day, 54-km ski march (SKI). Energy intake (kcal·day−1) increased (P < 0.01) from (mean ± SD (95% confidence interval)) 3098 ± 236 (2985, 3212) during MTT to 3461 ± 586 (3178, 3743) during SKI, while protein (g·kg−1·day−1) intake remained constant (MTT, 1.59 ± 0.33 (1.51, 1.66); and SKI, 1.71 ± 0.55 (1.58, 1.85)). Energy expenditure increased (P < 0.05) during SKI (6851 ± 562 (6580, 7122)) compared with MTT (5480 ± 389 (5293, 5668)) and exceeded energy intake. Protein flux, synthesis, and breakdown were all increased (P < 0.05) 24%, 18%, and 27%, respectively, during SKI compared with baseline and MTT. Whole-body protein balance was lower (P < 0.05) during SKI (–1.41 ± 1.11 (–1.98, –0.84) g·kg−1·10 h) than MTT and baseline. Muscle damage and soreness increased and performance decreased progressively (P < 0.05). The physiological consequences observed during short-term winter military training provide the basis for future studies to evaluate nutritional strategies that attenuate protein loss and sustain performance during severe energy deficits. Key words: nitrogen, dietary protein, recommended dietary allowance, stress. Résumé : Les conséquences des opérations militaires en hiver ne sont pas bien connues. Cette étude examine des soldats norvégiens (n = 21 hommes) qui participent a` un programme d’entraînement exigeant sur le plan physique afin d’évaluer si un programme d’entraînement militaire a` court terme modifie l’équilibre énergétique, le bilan protéique global, les lésions et les douleurs musculaires ainsi que la performance. Tous les jours, on évalue l’apport énergétique et la dépense d’énergie (D218O). De plus, on évalue le renouvellement global des protéines en condition postabsorptive ([15N]-glycine), les lésions et les douleurs musculaires ainsi que la performance (saut vertical) au début du programme, après 4 journées d’entraînement militaire (« MTT ») et après 3 autres journées comprenant 54 km de marche en ski (« SKI »). L’apport énergétique (kcal·jour–1) augmente (P < 0,01) de (moyenne ± écart-type (intervalle de confiance 95 %)) 3098 ± 236 (2985, 3212) au cours du MTT a` 3,461 ± 586 (3178, 3743) au cours de SKI, mais l’apport protéique (g·kg–1·jour–1) demeure constant (MTT, 1,59 ± 0,33 (1,51, 1,66) et SKI, 1,71 ± 0,55 (1,58, 1,85)). La dépense énergétique augmente (P < 0,05) au cours de SKI (6851 ± 562 (6580, 7122)) comparativement a` MTT (5480 ± 389 (5293, 5668)) et dépasse l’apport énergétique. Le flux protéique, sa synthèse et sa dégradation augmentent tous (P < 0,05) de 24 %, 18 % et 27 %, respectivement au cours de SKI comparativement aux valeurs initiales et a` MTT. Le bilan protéique global est plus faible (P < 0,05) au cours de SKI (–1,41 ± 1,11 (–1,98, –0,84) g·kg–1·10 h) comparativement a` MTT et aux valeurs initiales. Les lésions musculaires et la douleur s’accroissent et la performance diminue progressivement (P < 0,05). Les conséquences physiologiques observées au cours du programme d’entraînement militaire a` court terme en hiver constituent la base d’études ultérieures pour évaluer les stratégies nutritionnelles minimisant la perte de protéines et maintenant la performance durant des périodes de déficit énergétique profond. [Traduit par la Rédaction] Mots-clés : azote, protéines alimentaires, apport nutritionnel recommandé, stress.
Introduction During military combat and training operations daily energy expenditures typically are high because of long periods of low- to moderate-intensity physical activity and limited time for sleep or
rest (Tharion et al. 2005). Daily energy expenditures of military personnel during these conditions are similar to those achieved by athletes participating in prolonged sporting events and intense physical training programs (Loucks 2004; Black et al. 2012). While
Received 9 June 2014. Accepted 29 August 2014. L.M. Margolis, N.E. Murphy, S.M. McGraw, A.J. Young, S.J. Montain, and S.M. Pasiakos. Military Nutrition Division, US Army Research Institute of Environmental Medicine, 15 Kansas Street, Bldg. 42, Natick, MA 01760, USA. S. Martini, I. Thrane, J.-M. Blatny, and Y. Gundersen. Norwegian Defence Research Establishment, Instituttvn 20, N-2007 Kjeller, Norway. M.G. Spitz and J.W. Castellani. Thermal Mountain and Medicine Division, US Army Research Institute of Environmental Medicine, 15 Kansas Street, Bldg. 42, Natick, MA 01760, USA. J.C. Rood. Pennington Biomedical Research Center, Louisiana State University System, 6400 Perkins Rd., Baton Rouge, LA 70808, USA. Corresponding author: Stefan M. Pasiakos (e-mail: [email protected]). Appl. Physiol. Nutr. Metab. 39: 1395–1401 (2014) dx.doi.org/10.1139/apnm-2014-0212
Published at www.nrcresearchpress.com/apnm on 3 September 2014.
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athletes and other physically active individuals typically consume adequate dietary energy and protein to maintain energy balance and prevent decrements in protein balance, military personnel eat only as time permits during training and combat operations, and they generally fail to match energy expenditures with increased energy intake under those conditions (Montain and Young 2003). Consequently, such energy deficits may diminish whole-body protein balance and result in a loss of total body and lean body mass (Friedl et al. 2000; Montain and Young 2003; Tharion et al. 2005; Carbone et al. 2012; Margolis et al. 2013), which may, in part, contribute to increased muscle damage and decrements in physical performance (Knapik et al. 1987; Johnson et al. 1994; Friedl 1995; Nindl et al. 2007). Energy status is a primary regulatory of whole-body protein balance, as energy deficits generally result in negative whole-body protein balance (Young et al. 1991; Carbone et al. 2012). As such, identifying nutrition strategies that minimize the effects of energy deficit on protein retention has been a primary objective of our laboratory. We have demonstrated that consuming dietary protein at levels twice the recommended dietary allowance (RDA) promotes nitrogen retention and spares lean mass without decreasing calcium retention during sustained, moderate energy deficit (⬃1000 kcal·day−1) (Pikosky et al. 2008; Pasiakos et al. 2013b; Cao et al. 2014). Furthermore, we showed that consuming dietary protein at levels beyond twice the RDA (2× RDA 1.6 g·kg−1·day−1) provided no additional protection for nitrogen retention and lean body mass during moderate energy deficiency (Pasiakos et al. 2014). These controlled laboratory studies yielded critical insight regarding protein requirements for military personnel. However, whether or not our findings are generalizable to real-world operational conditions is unclear, as the degree of energy deficit experienced by military personnel during actual combat and training operations is often more severe than those imposed in laboratory studies. In addition, there have been no studies demonstrating the effects of real-world military operations on kinetic measures of whole-body protein balance. A review of various military training feeding programs found that a 7-day Norwegian winter military training program (WMT), where soldiers perform sustained physical activity and consume combat rations that provide ⬃150 g of dietary protein per day, would be a viable model to observe the extent to which short-term military operations modulate energy and whole-body protein balance. The purpose of this study was to evaluate energy and wholebody protein balance, and indices of muscle damage, soreness, and performance in Norwegian soldiers participating in a metabolically demanding, 2-phase training program (4 days, garrisonbased, military training task (MTT) and a 3-day ski march (SKI)). This was achieved by assessing and comparing energy expenditure, energy intake, whole-body protein balance, and measures of muscle damage, soreness, and physical performance during the 2 phases of training. We hypothesized that WMT would elicit an energy deficit resulting in a loss of total body mass, diminished whole-body protein balance, and increased biomarkers of muscle damage and scores of muscle soreness, culminating in decrements in physical performance. We expected the metabolic cost and the detrimental consequences of WMT to increase between MTT and SKI. We anticipated that findings from this observational study would provide the ecological basis to assess optimal nutritional strategies that offset the detrimental effects of real-world military operational stress.
Materials and methods Experimental design Norwegian conscripted soldiers (n = 21 males, age: 20 ± 1 years, height: 182 ± 7 cm, mass: 82 ± 9 kg), with minimal military experience (≤2 months) volunteered for this study after providing informed, written consent. The soldiers were scheduled to partic-
Appl. Physiol. Nutr. Metab. Vol. 39, 2014
ipate in this WMT (ambient temperature: –15 ± 4 °C (low –26 °C and high –6.2 °C)) as part of their typical training regimen. The training regimen before this WMT consisted of didactic classroom training, basic cold-weather military field training, and aerobictype physical training. This study was approved by the Human Use Review Committee at the US Army Research Institute of Environmental Medicine (Natick, Mass., USA) and the Regional Committees for Medical and Health Research Ethics (REK sør-øst, Oslo, Norway). Measures of energy expenditure and energy intake were obtained throughout WMT (Fig. 1). Whole-body protein turnover, measures of muscle soreness and damage, and physical performance were assessed at the beginning of training (baseline), following MTT, and immediately after completing SKI. MTT (days 1–4) consisted of weapons familiarization, mountainous terrain navigation, and general winter survival training. After completing the MTT data collection (morning day 5), soldiers began SKI (days 5–7), which comprised 6–10 h of skiing per day with ⬃50:10 min work to rest ratios. Soldiers travelled nearly 20 km per day while carrying ⬃45 kg of additional gear. Non-ski activities during SKI consisted of setting up winter camp, preparing meals, medical treatment and injury prevention, and preparation for the subsequent day. Energy balance: analysis of dietary intake and total daily energy expenditure Soldiers subsisted solely on Norwegian military arctic combat rations during WMT. As previously reported (McClung et al. 2013), soldiers were provided 3 rations/day (⬃3800 kcal·day−1) during MTT, and 4 rations/day (⬃5100 kcal·day−1) during SKI. Nutrient intake was determined from returned trash and ration food logs collected daily. Trained technicians verified ration food logs against returned wrappers and uneaten food items. Before the WMT began, soldiers consumed 3 meals per day at the local military dining facility, which provided a variety of healthy foods (e.g., milk, cereals, lean meats, fish, vegetables, and fruits). Energy expenditure assessments began the evening of day 0 using doubly labeled water (DLW) (DeLany et al. 1989). Soldiers provided a urine sample before consuming the DLW to determine background 18O and 2H enrichments. Soldiers fasted for a minimum of 4 h before consuming the isotope dose. Soldiers remained fasted for 8 h after ingesting the DLW (0.23 g H218O·kg total body water (TBW)−1 and 0.15 g 2H2O·kg TBW−1; Sigma–Aldrich, St. Louis, Mo., USA). Two soldiers were randomly chosen to consume local drinking water rather than DLW to control for changes in the natural abundance of 2H and 18O in local drinking water consumed (local water was analyzed independently to determine 2H and 18O enrichments). Rate of disappearance of 18O and 2H for soldiers dosed with DLW were corrected for mean changes in background enrichments based on control soldiers. Urine samples were collected daily. TBW was calculated by determining the regression line for the elimination of 2H and 18O and extrapolated to a maximum enrichment. Second-void morning urine samples were collected each morning during WMT. Enrichments of 2H and 18O were measured using isotope ratio mass spectroscopy (Finnigan Mat 252, Thermo Fisher Scientific, Waltham, Mass., USA). The 2H and 18O isotope elimination rates (kH and kO) were calculated by linear regression using the isotopic disappearance rates over the 7-day study to determine CO2 production according to Schoeller et al. (1986): rCO2 ⫽ (N/2.078)(1.01kO ⫺ 1.04kH) ⫺ 0.0246rH2Of where N is TBW; kO and kH are 18O and 2H isotope disappearance rates, respectively; and rH2Of is the rate of fractionated evaporated water loss and is estimated to be 1.05 N × (1.01 kO – 1.04 kH). Total daily energy expenditure was then calculated using the enPublished by NRC Research Press
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Fig. 1. Study design for this 2-phase 7-day winter military training program (4-day, garrison-based, military training task (MTT) and a 3-day ski march (SKI)). Study Days Baseline 1
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N-glycine Body Weight Performance Blood Draw
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ergy equivalent of CO2 for a respiratory quotient of 0.86 (Wolfe 2005). Although the respiratory quotient is an average, and measurements of substrate utilization were not performed, we have effectively applied this value in previous investigations where energy deficits were observed (Margolis et al. 2013). Energy balance for MTT and SKI were calculated by subtracting mean energy intake from mean energy expenditure for both study periods. Body mass was measured to the nearest 0.1 kg using a calibrated digital scale (Spider 2, Mettler Toledo, Mettler-Toledo GmbH, Germany) at baseline, MTT, and SKI to examine the effects of WMT on body mass. Body mass measurements were obtained in garrison for baseline and MTT and in the field after SKI. Measurements were conducted by the same study team member with participants wearing standardized clothing (undershirt, uniform pants, and socks) in the fasted state at the same time in the mornings of days 1, 5, and 8. Whole-body protein turnover Postabsorptive whole-body protein turnover was assessed by ingestion of [15N]-glycine as described by Waterlow et al. (1978). This end-product method was used because of its ability to estimate whole-body protein turnover noninvasively in free-living individuals (Bolster et al. 2001; Hartman et al. 2006). Total nitrogen ([15N]-nitrogen) enrichment was used to measure whole-body protein turnover to minimize bias of enrichment partitioning between the 2 metabolic pools (ammonia and urea) (Stein et al. 1986). After providing a urine sample to determine background [15N] enrichments, soldiers consumed a single dose of [15N]-glycine (2 mg·kg−1, Cambridge Isotope Laboratories, Andover, Mass., USA) at ⬃1900 h on days 1 (baseline), 4 (MTT), and 7 (SKI) following the final meal of the day. Urine was collected for the next 10 h, ending with the first-void of the following morning. Soldiers refrained from physical activity and consumption of food or energycontaining beverages during the 10-h collection period. Enrichments (i.e., ratio of tracer to tracee, tr:T) for [15N]-nitrogen was determined using isotope ratio mass spectroscopy (Metabolic Solutions Inc., Nashua, N.H., USA). Whole-body protein flux (Q), protein synthesis (PS), protein breakdown (PB), and net protein balance (NET) were calculated as follows: Q ⫽ d/corrected tr:T/10 × body mass × 24 PS ⫽ Q ⫺ (E/10 × body mass) × 6.25 × 24 PB ⫽ (Q ⫺ I/10 × body mass) × 6.25 × 24 NET ⫽ PS ⫺ PB
where Q is in gN·kg–1·day–1, and PS, PB, and NET are in g·kg–1·day–1; d denotes the oral dose of [15N] (g glycine × 0.1972); E is 10 h urinary nitrogen excretion; and I represents nitrogen intake of evening meal consumed prior to isotope dosing. Analyses of muscle damage, soreness, and performance Serum was isolated from blood samples collected after an overnight fast at baseline, MTT, and SKI to assess surrogate markers of muscle damage, including creatine kinase, lactate dehydrogenase (Beckman Coulter DXC 600 Pro, Beckman Coulter, Brea, Calif., USA), and myoglobin (Siemens Immulite 2000, Siemens Medical Solutions USA Inc., Malvern, Pa., USA). Corresponding subjective ratings of muscle soreness (deltoids, quadriceps, gluteus, and gastrocnemius/soleus) were collected from participants using a validated visual analogue scale; results were reported as a percentage, with higher scores indicating greater soreness (Montain et al. 2000). Peak power was measured at baseline, MTT, and SKI using the vertical jump test (Vertec, Jump USA, Sunnyvale, Calif., USA). After recording maximum reach height with heels flat on the ground, soldiers were given 3 jumps to attain maximal jump height. Attempts were separated by 1 min of recovery. The vertical jump test has previously been shown to be a valid field-expedient method to determine lower-body power output in a military population (Nindl et al. 2007). Vertical displacement (jump height) was calculated as the difference between maximal jump height and reach height and peak power was calculated using the following equation (Sayers et al. 1999): peak power ⫽ (60.7 × jump height) ⫹ (45.3 × body mass) ⫺ 2055
where peak power is in watts, jump height is in cm, and body mass is in kg. Statistical analysis Normality was confirmed using Shapiro–Wilk tests for dependent variables. Paired t tests were used to determine differences in energy expenditure, energy intake, and energy balance from MTT to SKI. Repeated measures ANOVA were used to evaluate changes over time (baseline vs. MTT vs. SKI) for all remaining dependent variables. Post hoc pairwise analyses were completed with Bonferroni corrections. Significance was set at P < 0.05. Data were analyzed using IBM SPSS Statistics for Windows (version 20.0. IBM Corp., Armonk, N.Y., USA) and expressed as means ± SD (95% confidence interval).
Results Energy balance Total daily energy expenditure during WMT was 6140 ± 394 (5950, 6330) kcal·day−1. Energy expenditure during SKI was 25% higher (P < 0.05) than MTT (Table 1). Independent of training phase, soldiers were in severe energy deficit (2899 ± 498 (2659, 3139) kcal·day−1) throughout WMT. Despite receiving an additional 1300 kcal·day−1 during SKI, energy deficit was 42% greater (P < 0.05) compared with MTT. Energy intake increased (362 ± 496 (123, 602) kcal·day−1, P < 0.05) during SKI; however, participants only consumed 66% of the energy provided during SKI compared with consuming 85% of the energy provided during MTT, with specific ration components regularly under consumed (Table 2). Protein and carbohydrate intake was similar (P > 0.05) between MTT and SKI, consuming ⬃200% RDA for protein and ⬃277% RDA for carbohydrate, while fat intake increased (P < 0.05) from MTT to SKI (Table 1). Body mass decreased (P < 0.05) 1.8 ± 1.0 (1.2, 2.4) kg from baseline (82.3 ± 9.7 (78.3, 86.3) kg) to MTT (80.5 ± 8.1 (76.8, 84.2) kg); however, remained unchanged (P > 0.05) from MTT to SKI (80.2 ± 7.9 (76.6, 83.8) kg). Published by NRC Research Press
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Table 1. Energy balance and relative macronutrient intake during a 7-day winter military training program. MTT
SKI
5480±389 (5293, 5668) 3098±236 (2985, 3212) −2382±499 (−2623, −2141)
6851±562 (6580, 7122)* 3461±586 (3178, 3743)* −3390±669 (−3713, −3068)*
−1
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Energy (kcal·day ) Energy expenditure Energy intake Energy balance
Macronutrient intake (g·kg−1day−1) Protein 1.59±0.33 (1.51, 1.66) Carbohydrate 4.42±0.85 (4.24, 4.61) Fat 1.51±0.29 (1.44, 1.57)
1.71±0.55 (1.58, 1.85) 4.74±1.62 (4.33, 5.15) 1.70±0.52 (1.57, 1.83)*
Note: Values for macronutrient intake are presented as means± SD (95% confidence interval); n = 19. MTT, military task training (days 1−4); SKI, ski march (days 5−7). Energy balance = intake − expenditure. *Different from MTT, P < 0.05.
Table 2. Ration item consumption during the 7-day winter military training program.
Entrées Chocolate bar Sweet oat biscuits Protein bar Beef jerky Bread Gummy fruit snack Dried cranberries Black currant jam Hot cocoa Energy drink Black currant tea Pâté Hot sauce Tuna fish
Consumed
Not consumed
94 95 93 90 85 84 82 74 65 60 50 48 43 37 34
6 5 7 10 15 16 18 26 35 40 50 52 57 63 66
Note: Percent ration component consumed from distributed Norwegian military arctic combat rations. Intake was determined from returned trash and ration food logs collected by research dietitians and trained study staff.
Whole-body protein turnover Whole-body Q increased over time (P < 0.05), as Q was 24% higher during SKI (2.16 ± 0.50 (1.90, 2.42) g N·kg−1· day−1) than baseline (1.70 ± 0.29 (1.55, 1.85) g N·kg−1· day−1) and MTT (1.74 ± 0.25 (1.61, 1.87) g N·kg−1· day−1; Fig. 2a). Q was similar (P > 0.05) between baseline and MTT. Similarly, there was no change (P > 0.05) in PS and PB from baseline to MTT, but PS and PB were upregulated (P < 0.05) by 18% and 27%, respectively, during SKI (Fig. 2b, 2c). Therefore, NET was maintained (P > 0.05) between baseline (0.06 ± 0.74 (−0.32, 0.44) g·kg−1·day−1) and MTT (–0.42 ± 1.08 (−0.98, 0.13) g·kg−1·day−1), but was lower (P < 0.05), and more negative, during SKI (–1.41 ± 1.11 (−1.98, –0.84) g·kg−1·day−1; Fig. 2d). Indices of muscle damage, soreness, and performance Creatine kinase and lactate dehydrogenase concentrations increased (P < 0.05) progressively from baseline, MTT, and SKI (Table 3). Myoglobin also increased (P < 0.05) during MTT and SKI, when levels averaged (mean increase) 40.8 ± 21.9 (28.4, 53.4) and 34.7 ± 21.8 (22.3, 47.1) ng·mL−1 higher than baseline, respectively. No difference in myoglobin (P > 0.05) was observed between MTT and SKI. Soldiers reported greater (P < 0.05) levels of muscle soreness at the end of both MTT and SKI compared with baseline (Table 3). Specifically, deltoid and quadriceps soreness rating increased (P < 0.05) progressively from baseline, MTT, and SKI. Gastrocnemius/soleus and gluteus soreness levels were higher (P < 0.05) during MTT and SKI compared with baseline, but remained steady (P > 0.05) during MTT to SKI. Lower body peak power (W) declined progressively (P < 0.05) from baseline (4870 ± 628 (4567, 5173)),
MTT (4736 ± 597 (4449, 5024)), and SKI (4537 ± 576 (4259, 4815)). Similarly, vertical jump height (cm) diminished over WMT with values declining (P < 0.05) from baseline (50.7 ± 5.6 (48.0, 53.3)), MTT (49.3 ± 5.3 (46.8, 51.9)), and SKI (46.3 ± 5.4 (43.6, 48.9)).
Discussion The primary findings of this longitudinal observational study were that this WMT program elicited severe energy deficits that likely contributed to a reduction in postabsorptive whole-body protein balance despite consuming dietary protein at levels approximately twice the RDA (Department of the Army 2001; Pasiakos et al. 2013a). Thus, our previous laboratory findings, which demonstrated that consuming protein at levels twice the RDA under controlled conditions promotes protein balance during short-term, moderate energy deficit (Pikosky et al. 2008; Pasiakos et al. 2013b), were not confirmed by the results of this field study. In addition to decrements in energy and whole-body protein balance, this WMT, which was conducted by soldiers with minimal military experience, caused significant muscle damage, muscle soreness, and diminished physical performance. Consistent with our hypothesis, energy expenditure was high during WMT. However, we did not anticipate that the energy expenditure levels achieved during WMT would be as high as we observed, particularly during the ski march (⬃6800 kcal·day−1). While energy expenditure values measured during SKI are extreme and likely unsustainable for prolonged periods, they are consistent with measurements reported by Hoyt et al. (1991), who observed comparable energy expenditures (⬃7100 kcal·day−1) during Marine Mountain Warfare Training. In that study, volunteers engaged in a 4-day period of prolonged training consisting of ski and snowshoe maneuvers. Given the level of activity, both marines performing mountain warfare training and Norwegian soldiers in the present study were in severe energy deficits (66% and 52%, respectively). Energy status is a primary regulator of whole-body protein balance (Young et al. 1991). During the early stages of energy deficit, whole-body protein breakdown and protein oxidation are typically upregulated to meet energy requirements, causing increased nitrogen excretion resulting in reductions in whole-body protein balance (Nair et al. 1987; Carbone et al. 2012). Findings from the current study are consistent with the interaction between energy and protein balance. Increasing dietary protein intake may be an effective nutritional countermeasure to minimize protein loss during severe energy deficit. Our laboratory has previously reported that consuming protein at levels twice the RDA spares whole-body protein balance during short-term (10 days) and sustained (21 days) moderate (⬃1000 kcal·day−1) energy deficit (Pikosky et al. 2008; Pasiakos et al. 2013b). When considering that soldiers often fail to consume adequate energy to match energy demands during military operations (Tharion et al. 2005; Nindl et al. 2007; Margolis et al. 2013), a recent consensus was developed that recommended that soldiers should consume 1.5–2.0 g protein·kg−1·day−1 to mitigate declines in Published by NRC Research Press
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Fig. 2. Whole-body protein flux (a), synthesis (b), breakdown (c), and balance (d) measured at baseline (day 1; empty bars), after a 4-day, garrison-based, military training task (MTT) (day 4; grey bars), and after a 3-day ski march (SKI) (day 7; black bars). Values are presented as means ± SD, n = 21. +, Different from MTT (P < 0.05); *, Different from baseline and MTT (P < 0.05).
b.
a.
14.0
*
+
12.0
2.0 g·kg-1·d-1
gN·kg-1·d-1
10.0 1.5 1.0
8.0 6.0 4.0
0.5
2.0 0.0
0.0 Baseline
MTT
SKI
c.
Baseline
MTT
SKI
Baseline
MTT
SKI
d. 14.0
0.5
*
12.0
0.0 g·kg-1·d-1
10.0 g·kg-1·d-1
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2.5
8.0 6.0
-0.5 -1.0
4.0
-1.5
2.0 0.0
*
-2.0 Baseline
MTT
SKI
Table 3. Markers of muscle damage and muscle soreness during the 7-day winter military training program. Baseline
MTT
SKI
Muscle damage Creatine kinase (IU·L−1) Lactate dehydrogenase (IU·L−1) Myoglobin (ng·mL−1)
265.5±156.6 (194.2, 336.7)a 140.0±15.1 (133.1, 146.9)a 34.8±7.1 (31.5, 38.0)a
837.3±539.7 (591.6, 1083.0)b 182.6±31.9 (168.1, 197.1)b 75.6±23.4 (65.0, 86.2)b
1209.6±753.1 (866.8, 1552.4)c 234.1±46.2 (213.1, 255.2)c 69.5±22.2 (59.4, 79.6)b
Muscle soreness Shoulders (%) Quadriceps (%) Gluteus (%) Calves (%)
9.3±9.8 (4.9, 13.8)a 5.4±10.1 (0.8, 10.0)a 3.4±5.7 (0.9, 6.0)a 7.5±9.4 (3.2, 11.7)a
33.3±21.2 (23.6, 43.0)b 21.21±18.6 (12.7, 29.7)b 19.7±19.3 (10.9, 28.4)b 20.9±16.8 (13.3, 28.6)b
45.3±26.7 (33.3, 57.2)c 34.3±27.8 (21.7, 50.0)c 29.8±29.1 (16.6, 43.1)b 32.5±27.1 (20.1, 44.8)b
Note: Values presented as means ± SD (95% confidence interval); n = 21. Means not sharing the same lowercase letter are different, P < 0.05. Baseline, day 1; MTT, military task training (days 1−4); SKI, ski march (days 5−7). Muscle soreness was determined using visual analog scale (i.e., % of a 100-mm line).
whole-body protein balance during periods of high metabolic demand (Pasiakos et al. 2013a). This study, which was the first to evaluate whole-body protein balance in response to a real-world military operation, demonstrated that despite dietary protein intakes within the recommended range (⬃1.7 g·kg−1·day−1), postabsorptive whole-body protein balance became progressively more negative during training, an effect largely attributed to the substantial energy deficit (⬃3400 kcal·day−1). It is important to recognize that the methodologies used in the current investigation differ from our previous work, which was conducted in laboratory settings (Pikosky et al. 2008; Pasiakos et al. 2013b). As such, comparing the results of the present study to our past investigations and drawing conclusions, specifically with regards to dietary composition, should be done cautiously. There are other methodological limitations that warrant consideration when interpreting the protein balance outcomes. First, the valid-
ity of the end-product has been questioned because of differences in [15N]-glycine administration (e.g., fasted, fed, exercise, resting, oral, and intravenous dosing) and the potential for metabolic bias to create differences in flux between urea and ammonia (Fern et al. 1985; Stein et al. 1986). We chose to measure whole-protein balance in the postabsorptive state under resting conditions (i.e., overnight) and report a single estimate of whole-body nitrogen balance to standardize any bias created by the method. We understand that basing conclusions on whole-body protein balance acquired in the postabsorptive state may also be considered tenuous, and that future studies should verify our conclusions by assessing protein turnover and resulting protein balance throughout the entire day (postabsorptive, postprandial, resting, and exercise conditions). However, we do believe the methods were applied appropriately given the austere conditions during which the measurements were made (Waterlow et al. 1978; Garlick et al. 1980), Published by NRC Research Press
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and that our findings, which demonstrate a postabsorptive loss of total body protein in response to metabolically demanding military training, are informative, reproducible, and valid (Duggleby and Waterlow 2005). Despite the severe energy deficit, particularly during SKI, we were unable to detect progressive declines in body mass, as volunteers only lost body mass during MTT. Because of expected logistical and operational constraints, only clothed body weights were obtained. As such, changes in the weight of clothing may have decreased our sensitivity to accurately assess changes in body mass. Alternatively, energy expenditure rates may have been overestimated (isotopic fractionation, loss of isotopes through respiration) during SKI, and energy intakes may have been underestimated, and contributed to an inflated estimate of energy deficit and expected weight loss. We also recognize that lacking body composition measurements limits our ability to evaluate decrements in protein balance in the context of lean body mass. Regardless, wholebody protein balance was more negative following SKI, which is consistent with an increase in energy expenditure and consequent energy deficit. The energy deficit was not deliberate, as is often the case in other military training programs. The energy provided, and thus energy consumed, during MTT and SKI was just not sufficient to match energy expenditure and particularly when food was left uneaten. During SKI, soldiers were provided with an additional ⬃1300 kcal·day−1, above what was provided during MTT, but only consumed an additional 300 kcal·day−1. It is important to recognize that the intent of the current study was not to manipulate typical dietary intake to match anticipated energy expenditure, but to observe the physiological responses of soldiers subsisting on rations according to standing Norwegian Army Command feeding policies for this specific training program. Under-consumption of combat rations by soldiers conducting military operations is not a new observation (Marriott 1995). Whether such under-consumption reflects suppression of ad libitum appetite, inability to consume certain types of ration components during operational conditions or simply soldier avoidance of certain ration components cannot be addressed by this study. However, soldiers in our study regularly avoided consuming the powdered-based beverage ration items that require rehydration (e.g., hot cocoa, black currant tea, and carbohydrate-based energy drinks). These items account for approximately 380 kcals per Norwegian military arctic combat ration. Considering that 4 rations were provided daily during SKI, soldiers not consuming any of these items would lose 1520 kcal per day of potential energy intake. We also observed that the majority of soldiers did not regularly consume the tuna fish (130 kcal, and 17 g protein) that was provided daily in their breakfast ration, and most avoided consuming other protein-containing items (pâté; 50 kcal and 2 g protein), which collectively amounted to 1850 kcal in unconsumed ration items (⬃50% of the measured energy deficit) and 25 g of protein each day during SKI. So while consumption of 100% of the food provided would not have prevented the entire energy deficit, more complete consumption of rations provided might have mitigated the level of energy deficit to levels comparable (⬃1000 kcal·day−1) with those used in previous laboratory investigations (Pikosky et al. 2008; Pasiakos et al. 2013b), where protein intakes twice the RDA did effectively offset decrements in protein balance. In the present study, physical performance (i.e., lower body peak power) declined progressively during WMT. Decrements in performance were observed in the presence of only modest reductions (2%) in body mass, whereas previous studies have suggested that body mass decrements of at least 5% to 10% are necessary before measurable declines in physical performance will be observed (Knapik et al. 1987; Johnson et al. 1994; Friedl 1995; Nindl et al. 2007). It is likely that the prolonged multi-stressor (extreme cold environment, sustained strenuous physical activity, and
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heavy load carriage) produced muscle damage, muscle soreness, and fatigue, which caused decrements in physical performance.
Conclusion In conclusion, soldiers participating in this WMT experienced severe energy deficits with increased muscle damage, muscle soreness, and declines in physical performance. More importantly, this is the first study to report decrements in postabsorptive whole-body protein balance in response to a military training program that produced severe energy deficits. The physiological decrements observed during this WMT make it an ideal study platform for future investigations to assess optimal nutritional strategies, such as energy dense, protein-containing eat-on-themove combat ration items for optimizing ration consumption to enhance resistance to whole-body protein loss and to promote physiological sustainment and recovery. Conflict of interest statement The authors declare no conflicts of interest.
Acknowledgements The authors thank the soldiers that participated in this research experiment, the command, and training staff at the Garrison in Sør-Varanger (Kirkenes, Norway) for their support of this study. The investigators adhered to the policies for protection of human subjects as prescribed in Army Regulation 70-25, and the research was conducted in adherence with the provisions of 32 CFR part 219. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. Any citations of commercial organizations and trade names in this report do not constitute an official Department of the Army endorsement of approval of the products or services of these organizations. This work was supported by the US Army Medical Research and Material Command and the Norwegian Defence Research Establishment, under agreement no. W81XWH-12-0279.
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ARTICLE Effects of winter military training on energy balance, whole-body protein balance, muscle damage, soreness, and physical performance Lee M. Margolis, Nancy E. Murphy, Svein Martini, Marissa G. Spitz, Ingjerd Thrane, Susan M. McGraw, Janet-Martha Blatny, John W. Castellani, Jennifer C. Rood, Andrew J. Young, Scott J. Montain, Yngvar Gundersen, and Stefan M. Pasiakos
Abstract: Physiological consequences of winter military operations are not well described. This study examined Norwegian soldiers (n = 21 males) participating in a physically demanding winter training program to evaluate whether short-term military training alters energy and whole-body protein balance, muscle damage, soreness, and performance. Energy expenditure (D218O) and intake were measured daily, and postabsorptive whole-body protein turnover ([15N]-glycine), muscle damage, soreness, and performance (vertical jump) were assessed at baseline, following a 4-day, military task training phase (MTT) and after a 3-day, 54-km ski march (SKI). Energy intake (kcal·day−1) increased (P < 0.01) from (mean ± SD (95% confidence interval)) 3098 ± 236 (2985, 3212) during MTT to 3461 ± 586 (3178, 3743) during SKI, while protein (g·kg−1·day−1) intake remained constant (MTT, 1.59 ± 0.33 (1.51, 1.66); and SKI, 1.71 ± 0.55 (1.58, 1.85)). Energy expenditure increased (P < 0.05) during SKI (6851 ± 562 (6580, 7122)) compared with MTT (5480 ± 389 (5293, 5668)) and exceeded energy intake. Protein flux, synthesis, and breakdown were all increased (P < 0.05) 24%, 18%, and 27%, respectively, during SKI compared with baseline and MTT. Whole-body protein balance was lower (P < 0.05) during SKI (–1.41 ± 1.11 (–1.98, –0.84) g·kg−1·10 h) than MTT and baseline. Muscle damage and soreness increased and performance decreased progressively (P < 0.05). The physiological consequences observed during short-term winter military training provide the basis for future studies to evaluate nutritional strategies that attenuate protein loss and sustain performance during severe energy deficits. Key words: nitrogen, dietary protein, recommended dietary allowance, stress. Résumé : Les conséquences des opérations militaires en hiver ne sont pas bien connues. Cette étude examine des soldats norvégiens (n = 21 hommes) qui participent a` un programme d’entraînement exigeant sur le plan physique afin d’évaluer si un programme d’entraînement militaire a` court terme modifie l’équilibre énergétique, le bilan protéique global, les lésions et les douleurs musculaires ainsi que la performance. Tous les jours, on évalue l’apport énergétique et la dépense d’énergie (D218O). De plus, on évalue le renouvellement global des protéines en condition postabsorptive ([15N]-glycine), les lésions et les douleurs musculaires ainsi que la performance (saut vertical) au début du programme, après 4 journées d’entraînement militaire (« MTT ») et après 3 autres journées comprenant 54 km de marche en ski (« SKI »). L’apport énergétique (kcal·jour–1) augmente (P < 0,01) de (moyenne ± écart-type (intervalle de confiance 95 %)) 3098 ± 236 (2985, 3212) au cours du MTT a` 3,461 ± 586 (3178, 3743) au cours de SKI, mais l’apport protéique (g·kg–1·jour–1) demeure constant (MTT, 1,59 ± 0,33 (1,51, 1,66) et SKI, 1,71 ± 0,55 (1,58, 1,85)). La dépense énergétique augmente (P < 0,05) au cours de SKI (6851 ± 562 (6580, 7122)) comparativement a` MTT (5480 ± 389 (5293, 5668)) et dépasse l’apport énergétique. Le flux protéique, sa synthèse et sa dégradation augmentent tous (P < 0,05) de 24 %, 18 % et 27 %, respectivement au cours de SKI comparativement aux valeurs initiales et a` MTT. Le bilan protéique global est plus faible (P < 0,05) au cours de SKI (–1,41 ± 1,11 (–1,98, –0,84) g·kg–1·10 h) comparativement a` MTT et aux valeurs initiales. Les lésions musculaires et la douleur s’accroissent et la performance diminue progressivement (P < 0,05). Les conséquences physiologiques observées au cours du programme d’entraînement militaire a` court terme en hiver constituent la base d’études ultérieures pour évaluer les stratégies nutritionnelles minimisant la perte de protéines et maintenant la performance durant des périodes de déficit énergétique profond. [Traduit par la Rédaction] Mots-clés : azote, protéines alimentaires, apport nutritionnel recommandé, stress.
Introduction During military combat and training operations daily energy expenditures typically are high because of long periods of low- to moderate-intensity physical activity and limited time for sleep or
rest (Tharion et al. 2005). Daily energy expenditures of military personnel during these conditions are similar to those achieved by athletes participating in prolonged sporting events and intense physical training programs (Loucks 2004; Black et al. 2012). While
Received 9 June 2014. Accepted 29 August 2014. L.M. Margolis, N.E. Murphy, S.M. McGraw, A.J. Young, S.J. Montain, and S.M. Pasiakos. Military Nutrition Division, US Army Research Institute of Environmental Medicine, 15 Kansas Street, Bldg. 42, Natick, MA 01760, USA. S. Martini, I. Thrane, J.-M. Blatny, and Y. Gundersen. Norwegian Defence Research Establishment, Instituttvn 20, N-2007 Kjeller, Norway. M.G. Spitz and J.W. Castellani. Thermal Mountain and Medicine Division, US Army Research Institute of Environmental Medicine, 15 Kansas Street, Bldg. 42, Natick, MA 01760, USA. J.C. Rood. Pennington Biomedical Research Center, Louisiana State University System, 6400 Perkins Rd., Baton Rouge, LA 70808, USA. Corresponding author: Stefan M. Pasiakos (e-mail: [email protected]). Appl. Physiol. Nutr. Metab. 39: 1395–1401 (2014) dx.doi.org/10.1139/apnm-2014-0212
Published at www.nrcresearchpress.com/apnm on 3 September 2014.
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athletes and other physically active individuals typically consume adequate dietary energy and protein to maintain energy balance and prevent decrements in protein balance, military personnel eat only as time permits during training and combat operations, and they generally fail to match energy expenditures with increased energy intake under those conditions (Montain and Young 2003). Consequently, such energy deficits may diminish whole-body protein balance and result in a loss of total body and lean body mass (Friedl et al. 2000; Montain and Young 2003; Tharion et al. 2005; Carbone et al. 2012; Margolis et al. 2013), which may, in part, contribute to increased muscle damage and decrements in physical performance (Knapik et al. 1987; Johnson et al. 1994; Friedl 1995; Nindl et al. 2007). Energy status is a primary regulatory of whole-body protein balance, as energy deficits generally result in negative whole-body protein balance (Young et al. 1991; Carbone et al. 2012). As such, identifying nutrition strategies that minimize the effects of energy deficit on protein retention has been a primary objective of our laboratory. We have demonstrated that consuming dietary protein at levels twice the recommended dietary allowance (RDA) promotes nitrogen retention and spares lean mass without decreasing calcium retention during sustained, moderate energy deficit (⬃1000 kcal·day−1) (Pikosky et al. 2008; Pasiakos et al. 2013b; Cao et al. 2014). Furthermore, we showed that consuming dietary protein at levels beyond twice the RDA (2× RDA 1.6 g·kg−1·day−1) provided no additional protection for nitrogen retention and lean body mass during moderate energy deficiency (Pasiakos et al. 2014). These controlled laboratory studies yielded critical insight regarding protein requirements for military personnel. However, whether or not our findings are generalizable to real-world operational conditions is unclear, as the degree of energy deficit experienced by military personnel during actual combat and training operations is often more severe than those imposed in laboratory studies. In addition, there have been no studies demonstrating the effects of real-world military operations on kinetic measures of whole-body protein balance. A review of various military training feeding programs found that a 7-day Norwegian winter military training program (WMT), where soldiers perform sustained physical activity and consume combat rations that provide ⬃150 g of dietary protein per day, would be a viable model to observe the extent to which short-term military operations modulate energy and whole-body protein balance. The purpose of this study was to evaluate energy and wholebody protein balance, and indices of muscle damage, soreness, and performance in Norwegian soldiers participating in a metabolically demanding, 2-phase training program (4 days, garrisonbased, military training task (MTT) and a 3-day ski march (SKI)). This was achieved by assessing and comparing energy expenditure, energy intake, whole-body protein balance, and measures of muscle damage, soreness, and physical performance during the 2 phases of training. We hypothesized that WMT would elicit an energy deficit resulting in a loss of total body mass, diminished whole-body protein balance, and increased biomarkers of muscle damage and scores of muscle soreness, culminating in decrements in physical performance. We expected the metabolic cost and the detrimental consequences of WMT to increase between MTT and SKI. We anticipated that findings from this observational study would provide the ecological basis to assess optimal nutritional strategies that offset the detrimental effects of real-world military operational stress.
Materials and methods Experimental design Norwegian conscripted soldiers (n = 21 males, age: 20 ± 1 years, height: 182 ± 7 cm, mass: 82 ± 9 kg), with minimal military experience (≤2 months) volunteered for this study after providing informed, written consent. The soldiers were scheduled to partic-
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ipate in this WMT (ambient temperature: –15 ± 4 °C (low –26 °C and high –6.2 °C)) as part of their typical training regimen. The training regimen before this WMT consisted of didactic classroom training, basic cold-weather military field training, and aerobictype physical training. This study was approved by the Human Use Review Committee at the US Army Research Institute of Environmental Medicine (Natick, Mass., USA) and the Regional Committees for Medical and Health Research Ethics (REK sør-øst, Oslo, Norway). Measures of energy expenditure and energy intake were obtained throughout WMT (Fig. 1). Whole-body protein turnover, measures of muscle soreness and damage, and physical performance were assessed at the beginning of training (baseline), following MTT, and immediately after completing SKI. MTT (days 1–4) consisted of weapons familiarization, mountainous terrain navigation, and general winter survival training. After completing the MTT data collection (morning day 5), soldiers began SKI (days 5–7), which comprised 6–10 h of skiing per day with ⬃50:10 min work to rest ratios. Soldiers travelled nearly 20 km per day while carrying ⬃45 kg of additional gear. Non-ski activities during SKI consisted of setting up winter camp, preparing meals, medical treatment and injury prevention, and preparation for the subsequent day. Energy balance: analysis of dietary intake and total daily energy expenditure Soldiers subsisted solely on Norwegian military arctic combat rations during WMT. As previously reported (McClung et al. 2013), soldiers were provided 3 rations/day (⬃3800 kcal·day−1) during MTT, and 4 rations/day (⬃5100 kcal·day−1) during SKI. Nutrient intake was determined from returned trash and ration food logs collected daily. Trained technicians verified ration food logs against returned wrappers and uneaten food items. Before the WMT began, soldiers consumed 3 meals per day at the local military dining facility, which provided a variety of healthy foods (e.g., milk, cereals, lean meats, fish, vegetables, and fruits). Energy expenditure assessments began the evening of day 0 using doubly labeled water (DLW) (DeLany et al. 1989). Soldiers provided a urine sample before consuming the DLW to determine background 18O and 2H enrichments. Soldiers fasted for a minimum of 4 h before consuming the isotope dose. Soldiers remained fasted for 8 h after ingesting the DLW (0.23 g H218O·kg total body water (TBW)−1 and 0.15 g 2H2O·kg TBW−1; Sigma–Aldrich, St. Louis, Mo., USA). Two soldiers were randomly chosen to consume local drinking water rather than DLW to control for changes in the natural abundance of 2H and 18O in local drinking water consumed (local water was analyzed independently to determine 2H and 18O enrichments). Rate of disappearance of 18O and 2H for soldiers dosed with DLW were corrected for mean changes in background enrichments based on control soldiers. Urine samples were collected daily. TBW was calculated by determining the regression line for the elimination of 2H and 18O and extrapolated to a maximum enrichment. Second-void morning urine samples were collected each morning during WMT. Enrichments of 2H and 18O were measured using isotope ratio mass spectroscopy (Finnigan Mat 252, Thermo Fisher Scientific, Waltham, Mass., USA). The 2H and 18O isotope elimination rates (kH and kO) were calculated by linear regression using the isotopic disappearance rates over the 7-day study to determine CO2 production according to Schoeller et al. (1986): rCO2 ⫽ (N/2.078)(1.01kO ⫺ 1.04kH) ⫺ 0.0246rH2Of where N is TBW; kO and kH are 18O and 2H isotope disappearance rates, respectively; and rH2Of is the rate of fractionated evaporated water loss and is estimated to be 1.05 N × (1.01 kO – 1.04 kH). Total daily energy expenditure was then calculated using the enPublished by NRC Research Press
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Fig. 1. Study design for this 2-phase 7-day winter military training program (4-day, garrison-based, military training task (MTT) and a 3-day ski march (SKI)). Study Days Baseline 1
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SKI 8
7 15
N-glycine Body Weight Performance Blood Draw
Morning Urine Collection Daily Ration Collection
ergy equivalent of CO2 for a respiratory quotient of 0.86 (Wolfe 2005). Although the respiratory quotient is an average, and measurements of substrate utilization were not performed, we have effectively applied this value in previous investigations where energy deficits were observed (Margolis et al. 2013). Energy balance for MTT and SKI were calculated by subtracting mean energy intake from mean energy expenditure for both study periods. Body mass was measured to the nearest 0.1 kg using a calibrated digital scale (Spider 2, Mettler Toledo, Mettler-Toledo GmbH, Germany) at baseline, MTT, and SKI to examine the effects of WMT on body mass. Body mass measurements were obtained in garrison for baseline and MTT and in the field after SKI. Measurements were conducted by the same study team member with participants wearing standardized clothing (undershirt, uniform pants, and socks) in the fasted state at the same time in the mornings of days 1, 5, and 8. Whole-body protein turnover Postabsorptive whole-body protein turnover was assessed by ingestion of [15N]-glycine as described by Waterlow et al. (1978). This end-product method was used because of its ability to estimate whole-body protein turnover noninvasively in free-living individuals (Bolster et al. 2001; Hartman et al. 2006). Total nitrogen ([15N]-nitrogen) enrichment was used to measure whole-body protein turnover to minimize bias of enrichment partitioning between the 2 metabolic pools (ammonia and urea) (Stein et al. 1986). After providing a urine sample to determine background [15N] enrichments, soldiers consumed a single dose of [15N]-glycine (2 mg·kg−1, Cambridge Isotope Laboratories, Andover, Mass., USA) at ⬃1900 h on days 1 (baseline), 4 (MTT), and 7 (SKI) following the final meal of the day. Urine was collected for the next 10 h, ending with the first-void of the following morning. Soldiers refrained from physical activity and consumption of food or energycontaining beverages during the 10-h collection period. Enrichments (i.e., ratio of tracer to tracee, tr:T) for [15N]-nitrogen was determined using isotope ratio mass spectroscopy (Metabolic Solutions Inc., Nashua, N.H., USA). Whole-body protein flux (Q), protein synthesis (PS), protein breakdown (PB), and net protein balance (NET) were calculated as follows: Q ⫽ d/corrected tr:T/10 × body mass × 24 PS ⫽ Q ⫺ (E/10 × body mass) × 6.25 × 24 PB ⫽ (Q ⫺ I/10 × body mass) × 6.25 × 24 NET ⫽ PS ⫺ PB
where Q is in gN·kg–1·day–1, and PS, PB, and NET are in g·kg–1·day–1; d denotes the oral dose of [15N] (g glycine × 0.1972); E is 10 h urinary nitrogen excretion; and I represents nitrogen intake of evening meal consumed prior to isotope dosing. Analyses of muscle damage, soreness, and performance Serum was isolated from blood samples collected after an overnight fast at baseline, MTT, and SKI to assess surrogate markers of muscle damage, including creatine kinase, lactate dehydrogenase (Beckman Coulter DXC 600 Pro, Beckman Coulter, Brea, Calif., USA), and myoglobin (Siemens Immulite 2000, Siemens Medical Solutions USA Inc., Malvern, Pa., USA). Corresponding subjective ratings of muscle soreness (deltoids, quadriceps, gluteus, and gastrocnemius/soleus) were collected from participants using a validated visual analogue scale; results were reported as a percentage, with higher scores indicating greater soreness (Montain et al. 2000). Peak power was measured at baseline, MTT, and SKI using the vertical jump test (Vertec, Jump USA, Sunnyvale, Calif., USA). After recording maximum reach height with heels flat on the ground, soldiers were given 3 jumps to attain maximal jump height. Attempts were separated by 1 min of recovery. The vertical jump test has previously been shown to be a valid field-expedient method to determine lower-body power output in a military population (Nindl et al. 2007). Vertical displacement (jump height) was calculated as the difference between maximal jump height and reach height and peak power was calculated using the following equation (Sayers et al. 1999): peak power ⫽ (60.7 × jump height) ⫹ (45.3 × body mass) ⫺ 2055
where peak power is in watts, jump height is in cm, and body mass is in kg. Statistical analysis Normality was confirmed using Shapiro–Wilk tests for dependent variables. Paired t tests were used to determine differences in energy expenditure, energy intake, and energy balance from MTT to SKI. Repeated measures ANOVA were used to evaluate changes over time (baseline vs. MTT vs. SKI) for all remaining dependent variables. Post hoc pairwise analyses were completed with Bonferroni corrections. Significance was set at P < 0.05. Data were analyzed using IBM SPSS Statistics for Windows (version 20.0. IBM Corp., Armonk, N.Y., USA) and expressed as means ± SD (95% confidence interval).
Results Energy balance Total daily energy expenditure during WMT was 6140 ± 394 (5950, 6330) kcal·day−1. Energy expenditure during SKI was 25% higher (P < 0.05) than MTT (Table 1). Independent of training phase, soldiers were in severe energy deficit (2899 ± 498 (2659, 3139) kcal·day−1) throughout WMT. Despite receiving an additional 1300 kcal·day−1 during SKI, energy deficit was 42% greater (P < 0.05) compared with MTT. Energy intake increased (362 ± 496 (123, 602) kcal·day−1, P < 0.05) during SKI; however, participants only consumed 66% of the energy provided during SKI compared with consuming 85% of the energy provided during MTT, with specific ration components regularly under consumed (Table 2). Protein and carbohydrate intake was similar (P > 0.05) between MTT and SKI, consuming ⬃200% RDA for protein and ⬃277% RDA for carbohydrate, while fat intake increased (P < 0.05) from MTT to SKI (Table 1). Body mass decreased (P < 0.05) 1.8 ± 1.0 (1.2, 2.4) kg from baseline (82.3 ± 9.7 (78.3, 86.3) kg) to MTT (80.5 ± 8.1 (76.8, 84.2) kg); however, remained unchanged (P > 0.05) from MTT to SKI (80.2 ± 7.9 (76.6, 83.8) kg). Published by NRC Research Press
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Table 1. Energy balance and relative macronutrient intake during a 7-day winter military training program. MTT
SKI
5480±389 (5293, 5668) 3098±236 (2985, 3212) −2382±499 (−2623, −2141)
6851±562 (6580, 7122)* 3461±586 (3178, 3743)* −3390±669 (−3713, −3068)*
−1
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Energy (kcal·day ) Energy expenditure Energy intake Energy balance
Macronutrient intake (g·kg−1day−1) Protein 1.59±0.33 (1.51, 1.66) Carbohydrate 4.42±0.85 (4.24, 4.61) Fat 1.51±0.29 (1.44, 1.57)
1.71±0.55 (1.58, 1.85) 4.74±1.62 (4.33, 5.15) 1.70±0.52 (1.57, 1.83)*
Note: Values for macronutrient intake are presented as means± SD (95% confidence interval); n = 19. MTT, military task training (days 1−4); SKI, ski march (days 5−7). Energy balance = intake − expenditure. *Different from MTT, P < 0.05.
Table 2. Ration item consumption during the 7-day winter military training program.
Entrées Chocolate bar Sweet oat biscuits Protein bar Beef jerky Bread Gummy fruit snack Dried cranberries Black currant jam Hot cocoa Energy drink Black currant tea Pâté Hot sauce Tuna fish
Consumed
Not consumed
94 95 93 90 85 84 82 74 65 60 50 48 43 37 34
6 5 7 10 15 16 18 26 35 40 50 52 57 63 66
Note: Percent ration component consumed from distributed Norwegian military arctic combat rations. Intake was determined from returned trash and ration food logs collected by research dietitians and trained study staff.
Whole-body protein turnover Whole-body Q increased over time (P < 0.05), as Q was 24% higher during SKI (2.16 ± 0.50 (1.90, 2.42) g N·kg−1· day−1) than baseline (1.70 ± 0.29 (1.55, 1.85) g N·kg−1· day−1) and MTT (1.74 ± 0.25 (1.61, 1.87) g N·kg−1· day−1; Fig. 2a). Q was similar (P > 0.05) between baseline and MTT. Similarly, there was no change (P > 0.05) in PS and PB from baseline to MTT, but PS and PB were upregulated (P < 0.05) by 18% and 27%, respectively, during SKI (Fig. 2b, 2c). Therefore, NET was maintained (P > 0.05) between baseline (0.06 ± 0.74 (−0.32, 0.44) g·kg−1·day−1) and MTT (–0.42 ± 1.08 (−0.98, 0.13) g·kg−1·day−1), but was lower (P < 0.05), and more negative, during SKI (–1.41 ± 1.11 (−1.98, –0.84) g·kg−1·day−1; Fig. 2d). Indices of muscle damage, soreness, and performance Creatine kinase and lactate dehydrogenase concentrations increased (P < 0.05) progressively from baseline, MTT, and SKI (Table 3). Myoglobin also increased (P < 0.05) during MTT and SKI, when levels averaged (mean increase) 40.8 ± 21.9 (28.4, 53.4) and 34.7 ± 21.8 (22.3, 47.1) ng·mL−1 higher than baseline, respectively. No difference in myoglobin (P > 0.05) was observed between MTT and SKI. Soldiers reported greater (P < 0.05) levels of muscle soreness at the end of both MTT and SKI compared with baseline (Table 3). Specifically, deltoid and quadriceps soreness rating increased (P < 0.05) progressively from baseline, MTT, and SKI. Gastrocnemius/soleus and gluteus soreness levels were higher (P < 0.05) during MTT and SKI compared with baseline, but remained steady (P > 0.05) during MTT to SKI. Lower body peak power (W) declined progressively (P < 0.05) from baseline (4870 ± 628 (4567, 5173)),
MTT (4736 ± 597 (4449, 5024)), and SKI (4537 ± 576 (4259, 4815)). Similarly, vertical jump height (cm) diminished over WMT with values declining (P < 0.05) from baseline (50.7 ± 5.6 (48.0, 53.3)), MTT (49.3 ± 5.3 (46.8, 51.9)), and SKI (46.3 ± 5.4 (43.6, 48.9)).
Discussion The primary findings of this longitudinal observational study were that this WMT program elicited severe energy deficits that likely contributed to a reduction in postabsorptive whole-body protein balance despite consuming dietary protein at levels approximately twice the RDA (Department of the Army 2001; Pasiakos et al. 2013a). Thus, our previous laboratory findings, which demonstrated that consuming protein at levels twice the RDA under controlled conditions promotes protein balance during short-term, moderate energy deficit (Pikosky et al. 2008; Pasiakos et al. 2013b), were not confirmed by the results of this field study. In addition to decrements in energy and whole-body protein balance, this WMT, which was conducted by soldiers with minimal military experience, caused significant muscle damage, muscle soreness, and diminished physical performance. Consistent with our hypothesis, energy expenditure was high during WMT. However, we did not anticipate that the energy expenditure levels achieved during WMT would be as high as we observed, particularly during the ski march (⬃6800 kcal·day−1). While energy expenditure values measured during SKI are extreme and likely unsustainable for prolonged periods, they are consistent with measurements reported by Hoyt et al. (1991), who observed comparable energy expenditures (⬃7100 kcal·day−1) during Marine Mountain Warfare Training. In that study, volunteers engaged in a 4-day period of prolonged training consisting of ski and snowshoe maneuvers. Given the level of activity, both marines performing mountain warfare training and Norwegian soldiers in the present study were in severe energy deficits (66% and 52%, respectively). Energy status is a primary regulator of whole-body protein balance (Young et al. 1991). During the early stages of energy deficit, whole-body protein breakdown and protein oxidation are typically upregulated to meet energy requirements, causing increased nitrogen excretion resulting in reductions in whole-body protein balance (Nair et al. 1987; Carbone et al. 2012). Findings from the current study are consistent with the interaction between energy and protein balance. Increasing dietary protein intake may be an effective nutritional countermeasure to minimize protein loss during severe energy deficit. Our laboratory has previously reported that consuming protein at levels twice the RDA spares whole-body protein balance during short-term (10 days) and sustained (21 days) moderate (⬃1000 kcal·day−1) energy deficit (Pikosky et al. 2008; Pasiakos et al. 2013b). When considering that soldiers often fail to consume adequate energy to match energy demands during military operations (Tharion et al. 2005; Nindl et al. 2007; Margolis et al. 2013), a recent consensus was developed that recommended that soldiers should consume 1.5–2.0 g protein·kg−1·day−1 to mitigate declines in Published by NRC Research Press
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Fig. 2. Whole-body protein flux (a), synthesis (b), breakdown (c), and balance (d) measured at baseline (day 1; empty bars), after a 4-day, garrison-based, military training task (MTT) (day 4; grey bars), and after a 3-day ski march (SKI) (day 7; black bars). Values are presented as means ± SD, n = 21. +, Different from MTT (P < 0.05); *, Different from baseline and MTT (P < 0.05).
b.
a.
14.0
*
+
12.0
2.0 g·kg-1·d-1
gN·kg-1·d-1
10.0 1.5 1.0
8.0 6.0 4.0
0.5
2.0 0.0
0.0 Baseline
MTT
SKI
c.
Baseline
MTT
SKI
Baseline
MTT
SKI
d. 14.0
0.5
*
12.0
0.0 g·kg-1·d-1
10.0 g·kg-1·d-1
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2.5
8.0 6.0
-0.5 -1.0
4.0
-1.5
2.0 0.0
*
-2.0 Baseline
MTT
SKI
Table 3. Markers of muscle damage and muscle soreness during the 7-day winter military training program. Baseline
MTT
SKI
Muscle damage Creatine kinase (IU·L−1) Lactate dehydrogenase (IU·L−1) Myoglobin (ng·mL−1)
265.5±156.6 (194.2, 336.7)a 140.0±15.1 (133.1, 146.9)a 34.8±7.1 (31.5, 38.0)a
837.3±539.7 (591.6, 1083.0)b 182.6±31.9 (168.1, 197.1)b 75.6±23.4 (65.0, 86.2)b
1209.6±753.1 (866.8, 1552.4)c 234.1±46.2 (213.1, 255.2)c 69.5±22.2 (59.4, 79.6)b
Muscle soreness Shoulders (%) Quadriceps (%) Gluteus (%) Calves (%)
9.3±9.8 (4.9, 13.8)a 5.4±10.1 (0.8, 10.0)a 3.4±5.7 (0.9, 6.0)a 7.5±9.4 (3.2, 11.7)a
33.3±21.2 (23.6, 43.0)b 21.21±18.6 (12.7, 29.7)b 19.7±19.3 (10.9, 28.4)b 20.9±16.8 (13.3, 28.6)b
45.3±26.7 (33.3, 57.2)c 34.3±27.8 (21.7, 50.0)c 29.8±29.1 (16.6, 43.1)b 32.5±27.1 (20.1, 44.8)b
Note: Values presented as means ± SD (95% confidence interval); n = 21. Means not sharing the same lowercase letter are different, P < 0.05. Baseline, day 1; MTT, military task training (days 1−4); SKI, ski march (days 5−7). Muscle soreness was determined using visual analog scale (i.e., % of a 100-mm line).
whole-body protein balance during periods of high metabolic demand (Pasiakos et al. 2013a). This study, which was the first to evaluate whole-body protein balance in response to a real-world military operation, demonstrated that despite dietary protein intakes within the recommended range (⬃1.7 g·kg−1·day−1), postabsorptive whole-body protein balance became progressively more negative during training, an effect largely attributed to the substantial energy deficit (⬃3400 kcal·day−1). It is important to recognize that the methodologies used in the current investigation differ from our previous work, which was conducted in laboratory settings (Pikosky et al. 2008; Pasiakos et al. 2013b). As such, comparing the results of the present study to our past investigations and drawing conclusions, specifically with regards to dietary composition, should be done cautiously. There are other methodological limitations that warrant consideration when interpreting the protein balance outcomes. First, the valid-
ity of the end-product has been questioned because of differences in [15N]-glycine administration (e.g., fasted, fed, exercise, resting, oral, and intravenous dosing) and the potential for metabolic bias to create differences in flux between urea and ammonia (Fern et al. 1985; Stein et al. 1986). We chose to measure whole-protein balance in the postabsorptive state under resting conditions (i.e., overnight) and report a single estimate of whole-body nitrogen balance to standardize any bias created by the method. We understand that basing conclusions on whole-body protein balance acquired in the postabsorptive state may also be considered tenuous, and that future studies should verify our conclusions by assessing protein turnover and resulting protein balance throughout the entire day (postabsorptive, postprandial, resting, and exercise conditions). However, we do believe the methods were applied appropriately given the austere conditions during which the measurements were made (Waterlow et al. 1978; Garlick et al. 1980), Published by NRC Research Press
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and that our findings, which demonstrate a postabsorptive loss of total body protein in response to metabolically demanding military training, are informative, reproducible, and valid (Duggleby and Waterlow 2005). Despite the severe energy deficit, particularly during SKI, we were unable to detect progressive declines in body mass, as volunteers only lost body mass during MTT. Because of expected logistical and operational constraints, only clothed body weights were obtained. As such, changes in the weight of clothing may have decreased our sensitivity to accurately assess changes in body mass. Alternatively, energy expenditure rates may have been overestimated (isotopic fractionation, loss of isotopes through respiration) during SKI, and energy intakes may have been underestimated, and contributed to an inflated estimate of energy deficit and expected weight loss. We also recognize that lacking body composition measurements limits our ability to evaluate decrements in protein balance in the context of lean body mass. Regardless, wholebody protein balance was more negative following SKI, which is consistent with an increase in energy expenditure and consequent energy deficit. The energy deficit was not deliberate, as is often the case in other military training programs. The energy provided, and thus energy consumed, during MTT and SKI was just not sufficient to match energy expenditure and particularly when food was left uneaten. During SKI, soldiers were provided with an additional ⬃1300 kcal·day−1, above what was provided during MTT, but only consumed an additional 300 kcal·day−1. It is important to recognize that the intent of the current study was not to manipulate typical dietary intake to match anticipated energy expenditure, but to observe the physiological responses of soldiers subsisting on rations according to standing Norwegian Army Command feeding policies for this specific training program. Under-consumption of combat rations by soldiers conducting military operations is not a new observation (Marriott 1995). Whether such under-consumption reflects suppression of ad libitum appetite, inability to consume certain types of ration components during operational conditions or simply soldier avoidance of certain ration components cannot be addressed by this study. However, soldiers in our study regularly avoided consuming the powdered-based beverage ration items that require rehydration (e.g., hot cocoa, black currant tea, and carbohydrate-based energy drinks). These items account for approximately 380 kcals per Norwegian military arctic combat ration. Considering that 4 rations were provided daily during SKI, soldiers not consuming any of these items would lose 1520 kcal per day of potential energy intake. We also observed that the majority of soldiers did not regularly consume the tuna fish (130 kcal, and 17 g protein) that was provided daily in their breakfast ration, and most avoided consuming other protein-containing items (pâté; 50 kcal and 2 g protein), which collectively amounted to 1850 kcal in unconsumed ration items (⬃50% of the measured energy deficit) and 25 g of protein each day during SKI. So while consumption of 100% of the food provided would not have prevented the entire energy deficit, more complete consumption of rations provided might have mitigated the level of energy deficit to levels comparable (⬃1000 kcal·day−1) with those used in previous laboratory investigations (Pikosky et al. 2008; Pasiakos et al. 2013b), where protein intakes twice the RDA did effectively offset decrements in protein balance. In the present study, physical performance (i.e., lower body peak power) declined progressively during WMT. Decrements in performance were observed in the presence of only modest reductions (2%) in body mass, whereas previous studies have suggested that body mass decrements of at least 5% to 10% are necessary before measurable declines in physical performance will be observed (Knapik et al. 1987; Johnson et al. 1994; Friedl 1995; Nindl et al. 2007). It is likely that the prolonged multi-stressor (extreme cold environment, sustained strenuous physical activity, and
Appl. Physiol. Nutr. Metab. Vol. 39, 2014
heavy load carriage) produced muscle damage, muscle soreness, and fatigue, which caused decrements in physical performance.
Conclusion In conclusion, soldiers participating in this WMT experienced severe energy deficits with increased muscle damage, muscle soreness, and declines in physical performance. More importantly, this is the first study to report decrements in postabsorptive whole-body protein balance in response to a military training program that produced severe energy deficits. The physiological decrements observed during this WMT make it an ideal study platform for future investigations to assess optimal nutritional strategies, such as energy dense, protein-containing eat-on-themove combat ration items for optimizing ration consumption to enhance resistance to whole-body protein loss and to promote physiological sustainment and recovery. Conflict of interest statement The authors declare no conflicts of interest.
Acknowledgements The authors thank the soldiers that participated in this research experiment, the command, and training staff at the Garrison in Sør-Varanger (Kirkenes, Norway) for their support of this study. The investigators adhered to the policies for protection of human subjects as prescribed in Army Regulation 70-25, and the research was conducted in adherence with the provisions of 32 CFR part 219. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. Any citations of commercial organizations and trade names in this report do not constitute an official Department of the Army endorsement of approval of the products or services of these organizations. This work was supported by the US Army Medical Research and Material Command and the Norwegian Defence Research Establishment, under agreement no. W81XWH-12-0279.
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