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Increased Protein Requirements in Female Athletes after Variable-Intensity Exercise


Medicine & Science in Sports & Exercise: November 2017 - Volume 49 - Issue 11 - p 2297–2304
doi: 10.1249/MSS.0000000000001366

Purpose Protein requirements are primarily studied in the context of resistance or endurance exercise with little research devoted to variable-intensity intermittent exercise characteristic of many team sports. Further, female populations are underrepresented in dietary sports science studies. We aimed to determine a dietary protein requirement in active females performing variable-intensity intermittent exercise using the indicator amino acid oxidation (IAAO) method. We hypothesized that these requirements would be greater than current IAAO-derived estimates in nonactive adult males.

Methods Six females (21.2 ± 0.8 yr, 68.8 ± 4.1 kg, 47.1 ± 1.2 mL O2·kg−1·min−1; mean ± SE) completed five to seven metabolic trials during the luteal phase of the menstrual cycle. Participants performed a modified Loughborough Intermittent Shuttle Test before consuming eight hourly mixed meals providing the test protein intake (0.2–2.66 g·kg−1·d−1), 6 g·kg−1·d−1 CHO and sufficient energy for resting and exercise-induced energy expenditure. Protein was provided as crystalline amino acid modeling egg protein with [13C]phenylalanine as the indicator amino acid. Phenylalanine turnover (Q) was determined from urinary [13C]phenylalanine enrichment. Breath 13CO2 excretion (F13CO2) was analyzed using mixed effects biphase linear regression with the breakpoint and upper 95% confidence interval approximating the estimated average requirement and recommended dietary allowance, respectively.

Results Protein intake had no effect on Q (68.7 ± 7.3 μmol·kg−1·h−1; mean ± SE). F13CO2 displayed a robust biphase response (R 2 = 0.66) with an estimated average requirement of 1.41 g·kg−1·d−1 and recommended dietary allowance of 1.71 g·kg−1·d−1.

Conclusions The protein requirement estimate of 1.41 and 1.71 g·kg−1·d−1 for females performing variable-intensity intermittent exercise is greater than the IAAO-derived estimates of adult males (0.93 and 1.2 g·kg−1·d−1) and at the upper range of the American College of Sports Medicine athlete recommendations (1.2–2.0 g·kg−1·d−1).

Supplemental digital content is available in the text.

1Faculty of Kinesiology and Physical Education, University of Toronto, Toronto, ON, CANADA; 2Frontier Research Laboratories, Institute for Innovation, Ajinomoto Co., Inc., Kawasaki, Kanagawa, JAPAN; 3Research Institute, Hospital for Sick Children, Toronto, ON, CANADA; 4Department of Nutritional Sciences, University of Toronto, Toronto, ON, CANADA; and 5Department of Pediatrics, University of Toronto, Toronto, ON, CANADA

Address for correspondence: Daniel R Moore, Ph.D., 100 Devonshire Place, Toronto, ON M5S 2C9, Canada; E-mail:

Submitted for publication March 2017.

Accepted for publication June 2017.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (

Dietary protein is important for athletes to replenish any exercise-induced losses and to provide the amino acid building blocks to remodel body and muscle proteins, both of which would ultimately support optimal postexercise recovery (30). Athletes participating in variable-intensity intermittent exercise characteristic of team sports (e.g., basketball, soccer, and ice hockey) engage in a unique activity pattern that could be considered to incorporate characteristics of both aerobic and resistance exercise. For example, these sports are aerobically based but also feature periods of high-intensity sprinting and rapid changes in velocity and direction (e.g., stop-and-go movements), the latter of which require high force production through the deceleration and acceleration phases, similar to dynamic strength or power exercises (34). It is well known that CHO serves as the primary energy source during high-intensity exercise (16). However, amino acid oxidation contributes up to approximately 5% of total energy during exercise but may increase to up to 10% during low glycogen conditions (23,35), which could occur during the later stages of variable-speed, high-intensity sports (19). The variable-intensity intermittent exercise pattern commonly observed in “stop-and-go” type sports can also increase markers of muscle damage and induce decrements in performance (19). Despite the importance of dietary protein for replenishing exercise-induced oxidative losses and supporting the repair and remodeling of body (and especially muscle) proteins after exercise (27), the effect of variable-intensity intermittent exercise on protein requirements, to our knowledge, has yet to be systematically investigated.

Protein requirements are generally suggested to be increased in athletes, as reflected by the current American College of Sports Medicine (ACSM) recommendation of 1.2–2.0 g·kg−1·d−1 (38). However, this broad recommendation may not accurately capture the specific needs of different athletes in light of their unique exercise demands. For example, bodybuilders are reported to require as little as 1.1 g·kg−1·d−1 (37) or as much as 2.2 g·kg−1·d−1 on a nontraining day (1), which presumably reflects the relative requirements of this population to support skeletal muscle remodeling, growth, and/or maintenance. By contrast, endurance athletes may require an intake of 1.2–1.8 g·kg−1·d−1 protein (17,25,35) to replace exercise-induced oxidative amino acid losses and to provide amino acids as precursors for the repair and/or remodeling of body proteins, both within skeletal muscle and throughout the body (27,35). Team sport athletes typically engage in exercise that has elements of both endurance and resistance exercise; however, to our knowledge, this population has never been the specific focus of a protein requirement study. Therefore, additional research would help refine athlete-specific recommendations for this large population. Furthermore, females are unfortunately underrepresented in sports science research (8), which is especially true of protein requirement studies that, to date, have been confined to males only (17,25,37). Although protein requirements have been retrospectively estimated in female athletes (14), we are aware of no studies that have had the primary objective of systematically investigating protein requirements in this population. Consequently, there is a need to define protein requirements in female athletes participating in team sports.

Our group previously used the minimally invasive indicator amino acid oxidation (IAAO) method to demonstrate that protein requirements in endurance athletes are about 50% greater than sedentary individuals (17), which is consistent with the generally greater recommendations for active individuals estimated using nitrogen balance (NBAL) methodology (29). However, NBAL determines the protein intake required to maintain nitrogen equilibrium, which, despite forming the primary basis of current consensus recommendations (38), has been argued to have little physiological relevance for an athlete (40). By contrast, the IAAO method identifies the protein requirement that maximizes whole body protein synthesis, which would be important for an athlete aiming to enhance recovery from, and potentially adaptation to, an exercise stimulus (29). Therefore, the objective of the current study is to prospectively investigate dietary protein needs of female athletes engaging in variable-intensity intermittent exercise using IAAO methodology. We hypothesized that protein requirements would be greater than the current recommendations for nonactive individuals determined by NBAL (0.8 g·kg−1·d−1) (42) and IAAO (1.2 g·kg−1·d−1) (15) but within ACSM recommendations for active populations (1.2–2.0 g·kg−1·d−1) (38).

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Ethics Statement

A verbal and written description of the study purpose, procedures, and potential risks was provided to all participants with an opportunity to ask questions before beginning the study. The study protocol was approved by the Research Ethics Board at the University of Toronto and conformed to the standards of the Declaration of Helsinki. Informed written consent was obtained from all participants before engaging in the study.

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Six healthy, active young adult females participated in the study Table 1. Four individuals were varsity athletes (three rowing and one ice hockey), one was a national level volleyball player, and one was a highly active recreational athlete. Participants were required to be healthy (PAR-Q+), have habitual activity levels of ≥45 min·d−1 on 5 d·wk−1 of moderate-vigorous physical activity (I-PAQ for adults 15–69 yr old), a predicted V˙O2max ≥44.6 mL O2·kg−1·min−1 (Leger Multistage Fitness Test) (22), no current use of hormonal contraceptives, and a predictable menstrual cycle (25–33 d) during the previous year as determined by interviews and the participants’ records of the 2–3 months before study enrollment.



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Study Protocol

Eligible participants were fitted with a SenseWear accelerometer armband (SWA; BodyMedia, Pittsburgh, PA) for 3-d wear to assess habitual caloric expenditure. On a separate day, participants visited the laboratory for body composition assessment (fat mass and fat-free mass [FFM]) using BodPod (Cosmed USA Inc., Chicago, IL) after avoiding food, water, and exercise for ≥4 h. Body composition was reassessed upon completion of all metabolic trials to account for potential changes over time (change in BW [kg] = −1.7 ± 0.62, change in FFM [kg] = −0.12 ± 0.36, change in body fat % = −1.8% ± 0.58%; mean ± SE). Average values from pre- and poststudy were used to normalize the data and are reported herein.

Each participant completed five to seven metabolic trials (n = 36) during the predicted luteal phase, which was defined as the second half of the menstrual cycle. Given that a 3-d isotope washout period is sufficient for the IAAO method (10), two metabolic trials within the same luteal phase were sometimes completed. After scheduling around participant’s training/competition requirements and/or menstrual cycle, the average duration to complete the study was 167 ± 91 d, which is similar to a previous study estimating lysine requirements across the menstrual cycle in nonexercising females (18). Participants consumed a 2-d adaptation diet containing 1.2 g·kg−1·d−1 of protein before each trial. This intake was selected to minimize metabolic variability during trial day (39) and to provide a level that was previously determined to be sufficient for nonexercising males by IAAO (15) and is within the recommended range for athletic populations (38). Adaptation diets contained sufficient energy to meet individual habitual daily caloric expenditure (Table 1), as determined by continuous 3-d accelerometer wear (SWA, BodyMedia).

Trial day methods were consistent with previous IAAO work in nonexercising populations (15) except for the addition of an exercise stimulus. Metabolic trials were performed on the same day as exercise to best capture the postexercise metabolic state. After an overnight fast, participants consumed a mixed-CHO beverage (1 g·kg−1 CHO, provided in a 1:1 mixture of maltodextrin [Polycal; Nutricia, Amsterdam, the Netherlands] and sports drink powder [Gatorade Thirst Quencher Powder; PepsiCo, Purchase, NY]) 30 min before arrival at the lab, in accordance with general preevent CHO guidelines (4). Participants then performed a modified version of the Loughborough Intermittent Shuttle Test, an exercise protocol that simulates the activity pattern of a soccer match (28) and was consistent with their habitual training and activity levels (Fig. 1). Briefly, the test involved four 15-min blocks in which a 17-m variable-intensity shuttle pattern was repeated 10 times with 5-min rests between each block. The entire test was 75 min in length, which also included a 5-min warm-up and cooldown at a self-selected pace. The running pace in our study was based on the percentage of average maximum speed obtained by the entire group of participants during the aerobic assessment (3.45 m·s−1) instead of the percentage of individual V˙O2max. In this way, the same audio prompt was used for all participants with the following 17-m shuttle paces: 1.7 m·s−1 (walk), 2.1 m·s−1 (jog), 3.1 m·s−1 (run), and an all-out sprint. The average energy expenditure during the Loughborough Intermittent Shuttle Test was 8.7 ± 0.2 kcal·kg−1 (range 8.5–9.1 kcal·kg−1, determined from accelerometer).



After exercise, participants consumed their first of eight hourly mixed meals containing the test protein intake (0.2–2.66 g·kg−1·d−1; Table 2) and sufficient energy and CHO content in the form of test beverages containing crystalline amino acids (Ajinomoto North America Inc., Raleigh NC), protein-free powder (PFD-1; Mead Johnson, Evansville, IN), fruit flavoring powder (Tang; Kraft, Don Mills, Canada), grapeseed oil, and maltodextrin (Polycal®) as well as protein-free cookies. Trial day energy intake was estimated as follows:



where RMR represents the resting metabolic rate determined from a 3-d average of accelerometer wear (SWA, BodyMedia), 1.6 represents a moderate activity factor, and 0.1429 kcal·kg−1·min−1 represents the estimated average energy cost of the exercise, from accelerometer data (SWA, BodyMedia) in a pilot study (n = 3).

The preexercise beverage (1 g·kg−1 CHO) and the daily CHO intake (6 g·kg−1·d−1 including the preexercise drink) were selected to meet current recommendations for this population (4). In addition, the sufficient energy intake would also minimize amino acid oxidation as an energy substrate, which permitted an accurate estimate of the minimum dietary protein requirement. The fifth meal contained a priming dose of NaH13CO2 (0.176 mg·kg−1) and L-[1-13C]phenylalanine (1.86 mg·kg−1; CIL Canada, Inc., Montreal, Canada). After the fifth meal, each hourly meal replaced 1.2 mg·kg−1 of unlabeled phenylalanine with L-[1-13C]phenylalanine to maintain an isotopic steady state and a constant dose of phenylalanine per meal (total phenylalanine content = 30.5 mg·kg−1·d−1). Tyrosine was provided in excess (40 mg·kg−1·d−1) to ensure the phenylalanine carboxyl carbon would be partitioned into either synthesis or oxidation (43). Protein intakes received by each subject are noted in Table 2, and the individual amino acid content of the diets was described previously (17).

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Sample Collection and Analysis

Breath samples

Three baseline breath samples and two baseline urine samples were collected at 15- and 30-min increments respectively, starting 15 min after the fourth meal and before ingestion of the L-[1-13C]phenylalanine tracer. Beginning 30 min after the seventh meal, breath (n = 5) and urine samples (n = 3) were collected at isotopic plateau according to the same frequency as baseline sampling. Urine samples were stored at −80°C until analysis. We performed time course pilot trials to confirm that metabolic (i.e., V˙CO2) and isotopic (i.e., 13CO2) steady state were achieved before baseline sampling (see Document, Supplemental Digital Content 1, Description of the pilot study and its results, V˙CO2 was measured over a 30-min period between the baseline and the plateau time points by indirect calorimetry (IX-TA-220; iWorx Systems, Inc., Dover, NH). Breath samples were collected using a system that permits the removal of dead space air (Easy-Sampler; QuinTron Instrument Company, Inc., Milwaukee, WI) into vacuumed Exertainer tubes (Labco, Ltd., Ceredigion, UK). Samples were analyzed for 13CO2 enrichment by continuous-flow ratio mass spectrometry for (CF_IRMS 20/20 isotope analyzer; PDZ Europa Ltd., Cheshire, UK). 13CO2 excretion (F13CO2; μmol·kg−1·h−1) was calculated as done previously (15,17).

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Urine samples

Urinary L-[1-13C]phenylalanine enrichment was determined by LC/MS/MS (API4000 triple quadrupole mass spectrometer; Applied Biosystems, Foster City, CA) in a positive electrospray ionization mode. Phenylalanine flux (PheRa, μmol·kg−1·h−1) and phenylalanine oxidation (PheOx; μmol·kg−1·h−1) were calculated as previously detailed (15,17).

Pregnanediol-3-glucuronide (PdG), the urinary metabolite of the luteal phase hormone progesterone (41), was quantified using a commercially available ELISA kit (Arbor Assays K037-H1; Arbor Assays, Ann Arbor, MI) and normalized to creatinine (BioAssay Systems cat. DICT-500; BioAssay Systems LLC, Hayward, CA). Urinary PdG concentrations from a sample in each metabolic trial (luteal phase; high expected PdG) was compared with PdG concentrations from a sample collected during the follicular phase (low expected PdG) in each participant to estimate whether individuals were in the luteal phase during metabolic trials.

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Statistical Analysis

Changes in PheRa, F13CO2, and PheOx were determined as a function of protein intake using a mixed linear model with the participant as a random variable using Proc Mixed program (SAS university version; SAS Institute Japan, Tokyo, Japan). Biphasic linear regression was applied to F13CO2 data to determine the breakpoint, which forms the basis of the IAAO method and represents the estimated average protein requirement (EAR). The protein requirement estimate was determined using F13CO2 rather than PheOx because F13CO2 is a more direct end point of intracellular metabolism and in this case is more reflective of whole body protein synthesis (32). In accordance with previous literature, a 95% confidence interval (CI) approximating the recommended dietary allowance (RDA) was calculated for this population (11,15,17). The use of the IAAO technique has been used extensively to estimate dietary indispensable amino acid and protein requirements with five to eight participants consuming either seven defined test protein intakes or seven unique test protein intakes (i.e., a total of ~35–56 metabolic trials) having a similar effect on the precision of the 95% CI (i.e., ranging from ~15% to 30% of the EAR) (11,15,18). Therefore, the present study included n = 6 participants with a target of seven unique intakes per participant (i.e., a total of 42 metabolic trials), which is a more robust approach to estimate requirements than providing seven defined intakes. Our 95% CI was approximately 21% of our EAR (see below) with n = 36 intakes, which compares favorably to previous studies that used N = 56 trials at seven defined intakes (i.e., ~30%) (15) or n = 56 random intakes (i.e., ~19%) (11). To determine whether the breakpoint determined herein (EAR1) differed from that of previously published nonexercising males (EAR2) (15), we first converted the 95% CI to SE (SE1 and SE2, respectively) by dividing the difference between the upper and the lower 95% CI of each EAR by 3.92. The extent of overlapping CI was determined by the following equation: (EAR1 – EAR2) ± 1.96 [√(SE1 2 + SE2 2)], whereby the null hypothesis was rejected if the interval did not contain zero.

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Phenylalanine flux

Average phenylalanine flux was 68.7 ± 17.3 μmol·kg−1·h−1, or 85.17 ± 17.3 μmol·kg (FFM)−1·h 1. The slope of PheRa against protein intake was not significantly different from zero (P = 0.54) (Fig. 2), indicating that protein intake did not affect phenylalanine flux. Thus, the metabolic pool of phenylalanine was consistent across trials, and any changes in phenylalanine oxidation can be attributed to alterations in whole-body protein synthesis (15).



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Estimated average and RDA for protein

The F13CO2 breakpoint after biphasic linear regression (R 2 = 0.66) revealed an EAR of 1.41 g·kg−1·d−1 and an upper 95% CI of the estimate (approximating the RDA) of 1.71 g·kg−1·d−1 (Fig. 3). The F13CO2 breakpoint determined in the present study was greater (P < 0.05) than that previously determined in nonexercising males (i.e., 0.93 g·kg−1·d−1 with an upper 95% CI of 1.24 g·kg−1·d−1) (15). The FFM-normalized F13CO2 (R 2 = 0.58) EAR and RDA were 1.69 g·kg (FFM)−1·d−1 and 2.24 g·kg (FFM)−1·d−1, respectively.



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Urinary PdG

Circulating progesterone concentrations are generally stable during the follicular phase, rising steadily during the luteal phase and reflected by a prominent peak in the urinary metabolite PdG (41). Metabolic trials took place during the estimated luteal phase based on menstrual cycle tracking. However, post hoc analysis revealed that creatinine-normalized urinary PdG levels from some metabolic trials were lower than samples from the same participants’ urinary PdG during the estimated follicular phase (n = 8), indicating that participants may not have been in the luteal phase during those trials. Reanalysis of the F13CO2 after removing those eight trials (n = 28; R 2 = 0.65) revealed a breakpoint of 1.52 g·kg−1·d−1 and RDA of 1.67 g·kg−1·d−1. Although this EAR was ~8% greater (P < 0.05) than when all trials were included, the biological significance of this small difference is unclear. Therefore, we would suggest the breakpoint analysis with all trials included would represent a more robust estimation of the protein requirements of the present population (i.e., ~80% of trials in the luteal phase).

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Population guidelines based on NBAL methodology do not consider that regular exercise has a modifiable effect on minimum protein requirements with the current RDA being set at 0.8 g·kg−1·d−1, which is 2 SD above the intake sufficient for 50% of the population (i.e., EAR) and designed to meet the needs of 97.5% of the population (42). These recommendations are at odds with current sports science consensus statements, suggesting a broad range of 1.2–2.0 g·kg−1·d−1 should be consumed by athletic populations (38). Given that studies primarily address the nutrient needs of males as well as the dichotomous endurance or strength athletes, the purpose of this study was to determine an EAR and RDA for protein in females engaged in team sportlike exercise. The present study is the first to determine a protein requirement by IAAO in an exercising female population and provide additional evidence that protein requirements in this active population are greater than the current minimum requirement. More specifically, our data reveal that performance of an acute bout of variable-intensity exercise results in an EAR of 1.41 g·kg−1·d−1 and an approximate RDA (i.e., sufficient for 95% of the population) and 1.71 g·kg−1·d−1. Thus, our results exceed the current recommendations of 0.8 g·kg−1·d−1 by NBAL (42) and 1.2 g·kg−1·d−1 by IAAO (15), demonstrating that variable-intensity exercise increases daily protein requirements in active females during the luteal phase.

Phenylalanine flux was numerically greater in our study compared with nonexercise males when normalized to body mass (~72 vs ~58 μmol·kg−1·h−1, respectively) and FFM (~85 vs ~72 μmol·kg (FFM)−1·h−1, respectively) (15), which is consistent with our previous observations in endurance-trained males on a day in which they trained (17) and in body builders on a rested day (1). Phenylalanine (30.5 mg·kg−1·d−1) was not limiting because its requirement in the presence of excess tyrosine is an estimated 9.1 mg·kg−1·d−1 (43), which suggests the greater flux in our study relative to sedentary males (15) at an identical phenylalanine intake would be the result of a greater whole body protein turnover. Although there may be slight fluctuations in basal metabolism across the menstrual cycle with peaks occurring during the luteal phase (3), we speculate that the greater phenylalanine flux in the present study relative to nonexercising adults (15) is due to the performance of the intermittent stop-and-go exercise stimulus and/or the trained nature of our participants. For example, muscle and whole body protein turnover are increased during recovery from acute exercise (2,9) as well as in the trained state at rest (31,36). Regardless of the physiological basis for this overall increased phenylalanine flux, there was no effect of protein intake on flux, which is a requirement for the robust estimation of protein recommendations by F13CO2 analysis (11,15).

Despite the importance of dietary protein to support optimal recovery, we are aware of no studies that have prospectively estimated protein requirements in females, which is unfortunately in line with the underrepresentation of this sex in sport and exercise science research (8). Indeed, current consensus recommendations, which are based almost exclusively on research in males, do not delineate potential sex-specific differences (38). Moreover, these recommendations are based primarily on the dichotomous exercise modalities of resistance and endurance exercise, which may ultimately reflect the generally broad, nonspecific nature of the current range of recommended intakes (i.e., 1.2–2.0 g·kg−1·d−1) (38). To address these sex and exercise modality-based gaps in the literature, we studied the effect of variably-intensity exercise on protein requirements in active females during the luteal phase. Our study used an exercise stimulus that is designed to mimic the activity pattern of a soccer match to model team sport activity in general (28). Given that most athletes train daily (5) and that aerobic exercise can increase the oxidative disposal of amino acids (35) and stimulate muscle protein synthesis (7), we studied our participants on a day in which they exercised as we speculate this would result in greater protein requirements than a nontraining day. Contrary to the current RDA of 0.8 g·kg−1·d−1, which was determined by NBAL and does not consider activity level as a major influencer of protein requirements (42), our study demonstrates that variable-intensity exercise increases protein requirements above this current recommendation. Although these differences may be related in part to the respective methodologies used (i.e., NBAL vs IAAO), comparison of our values to an adult population using a similar IAAO technique demonstrates that our active females had ~1.5-fold increase in EAR compared with nonexercising males (15). Our data are broadly aligned with previous research in males suggesting resistance and endurance exercise increase protein requirements (1,36). Interestingly, the ~75% increase in protein requirements for strength trained athletes (including football and rugby players) was previously suggested to be related to the incorporation of aerobic-based exercise (e.g., wind sprints) within the participants’ habitual sport-specific training (36). Therefore, in apparent agreement with their male counterparts (36), protein requirements in female athletes engaging in variable-intensity exercise are also increased relative to a nonexercising male population (15). Importantly, the requirement determined herein is toward the upper range of the broad requirements by the ACSM consensus statement of 1.2–2.0 g·kg−1·d−1 for athletes (38) and serves to provide a more refined athlete-specific recommendation for females engaged in team sports. The significance of this is highlighted by the tendency of these athletes to eat less than their male counterparts (33) and, in the case of female soccer players, to consume protein below (i.e., 1.2–1.4 g·kg−1·d−1) (12) the present recommendation.

It is becoming increasingly apparent that dietary protein requirements should be population-specific (38). Lysine requirements during the luteal phase in rested females are similar to that of males (18,44), suggesting the greater protein requirements determined herein relative to nonexercised males by IAAO may not be related to studying our female athletes during the luteal phase but rather to an interactive effect of the exercise stimulus. The approximate average energy expenditure during our exercise stimulus was 735 kcal, which could translate into ~9 g of protein (assuming a conservative 5% energy contribution) used as fuel during the exercise or a modest 0.13 g·kg−1·d−1 increase in daily requirement due to oxidative losses (23). It is possible that the amino acid contribution to fuel use may have increased during the latter stages of the variable-intensity exercise as muscle glycogen stores decreased (23,35), in which case 0.13 g·kg−1·d−1 could be interpreted as underestimate of the oxidative losses. Nevertheless, the potential oxidative amino acid losses in the present study may account for at least ~27% of the difference in protein requirements compared with nonexercised males (15). Inasmuch as oxidative amino acid losses contribute to the greater protein intake, novice athletes in the early stages of training may require slightly greater protein intakes due to a greater reliance on this macronutrient to support energy needs in the untrained state (24). Alternatively, the majority of the increased protein requirement relative to males at rest (15) may be attributable to the exercise-induced metabolic changes sustained throughout the postexercise period and/or training-induced changes in this population, such as the requirement to repair any exercised-induced muscle damage (27) and/or to support exercise-induced muscle or whole-body protein remodeling (30). In potential support, F13CO2 at plateau in the present study was lower than sedentary males (i.e., ~0.3 vs ~0.7 μmol·kg−1·h−1) (15), which would suggest that at an equivalent phenylalanine intake that whole body protein synthesis was greater in the present study. Further work is required to determine the contribution of the acute exercise bout per se to protein requirements versus the training status of our female population. However, athletes aiming to optimize their recovery from and adaptation to exercise would ostensibly benefit from maximizing whole body protein synthesis, which in our hands suggests an intake of 1.71 g·kg−1·d−1 would satisfy this goal.

There is some evidence that hormonal fluctuations across the menstrual cycle may lead to slight variations in protein metabolism and thus dietary needs across the cycle (6). Of relevance to active females, estrogen, which peaks in the late follicular phase and again during the luteal phase, has a protein sparing effect (13). By contrast, progesterone, which peaks only during the luteal phase, is generally considered to be catabolic in nature (20). Consequently, lysine requirements (18) and leucine flux and oxidation (21) are elevated during the luteal phase, which are likely related to the moderating effect of progesterone that would antagonize the potential protein-sparing effect of estrogen. We observed a greater urinary concentration of the progesterone-metabolite PdG (41) in the majority of trials, suggesting our athletes were exercising during a period of relatively greater progesterone compared with what would be expected during the follicular phase. Interestingly, there is no effect of menstrual phase (and presumably the associated changes in hormonal environment) on postexercise muscle protein synthesis (26), suggesting the protein requirement to optimize muscle remodeling would be similar in female athletes across the menstrual phase. Inasmuch as the greater protein requirement determined herein was influenced by the need to replace exercise-induced oxidative amino acid losses, it is possible that protein requirements for female athletes during the follicular phase may be slightly lower due to an estrogen-mediated attenuation in amino acid oxidation (13). Reanalysis of our data after removal of all low PdG (suggestive of exercise in the follicular phase or at least in the presence of low progesterone) resulted in an ~8% increase in protein requirements. Therefore, we speculate that the protein requirements determined in the current study represent the upper level of requirements for female athletes and would subsequently support the dietary need for this macronutrient across the entire menstrual cycle.

In summary, we used the IAAO method to identify a minimal dietary protein requirement for females who engage in team sportlike exercise. Our results suggest an RDA of 1.71 g·kg−1·d−1, which is higher than the current RDA (0.8 g·kg−1·d−1) and previous IAAO work in nonexercising males (1.2 g·kg−1·d−1) but generally consistent with ACSM Position Stand for protein requirements in active populations (i.e., 1.2–2.0 g·kg−1·d−1) (38). Although our RDA would represent ~14% energy from protein in our study, some reports suggest female team sport athletes may not reach the suggested intake when expressed relative to body weight (i.e., 1.71 g·kg−1·d−1) (5,12), especially if energy intake is suboptimal for their training needs (33). Therefore, our data highlight the need to further develop and refine population-specific protein requirements to ensure that both male and female athletes engaged in all exercise modalities meet the metabolic demand for this important macronutrient.

The authors thank the study participants for generously offering their time and effort. They also extend thanks to Mahroukh Radi and Mary-Ann Ryan of the Research Institute, Hospital for Sick Children, Toronto, for their invaluable expertise in sample analysis. This study was supported by an Ajinomoto Innovation Alliance Program Award to Daniel Moore.

The coauthor Hiroyuki Kato is employed by Ajinomoto Co. Inc. However, this relationship had no effect on study funding, and Ajinomoto Co. Inc. was not involved in any aspect of the study execution or analysis. Results of the present study do not constitute endorsement by the ACSM and are presented honestly without fabrication, falsification, or inappropriate manipulation of the data.

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