nutrients

Article
Dietary Intake of Protein and Essential Amino Acids for
Sustainable Muscle Development in Elite Male Athletes
Marius Baranauskas 1, * , Ingrida Kupčiūnaitė 1 and Rimantas Stukas 2

1 Faculty of Biomedical Sciences, Panevėžys University of Applied Sciences, 35200 Panevėžys, Lithuania;
[email protected]
2 Institute of Health Sciences, Faculty of Medicine, Department of Public Health, Vilnius University,
01513 Vilnius, Lithuania; [email protected]
* Correspondence: [email protected]

Abstract: Athletes need to develop a relatively high muscle mass and low body adipose tissue
for the sake of better athletic performance. A full range of nine essential amino acids and eleven
non-essential amino acids have to attend in appropriate amounts for protein biosynthesis. The aim
of the observational comparative cross-sectional study was to assess the association between the
diet quality profile and training-induced muscle mass estimated by bioelectrical impedance among
elite male athletes. The research sample comprised 18.1 ± 3.1 year-old Lithuanian professional male
athletes (n = 234). The study participants were enrolled to complete 24-h dietary recalls of three
non-consecutive days. The body composition was assessed using the bioelectrical impedance analysis
(BIA) method. The present study showed a significant insufficiency of the mean carbohydrate intake
of 5.7 g/kg/day in a group of aerobic male athletes. The lower muscle mass of aerobic male athletes
was related to the lower-carbohydrate diet (adjusted odd ratio (ORadj ) 0.3; 95% confidence interval
(CI): 0.1–0.7). The mean protein intake of 1.8 g/kg/day was optimal for anabolism in the samples of
both anaerobic and aerobic male athletes. The protein intake in appropriate doses was potentially
associated with an increase in muscle mass only in anaerobic male athletes (ORadj 2.2; 95% CI: 1.3–3.7).
The positive relationship was revealed between the possible muscle mass gain and the increased
intakes of amino acids such as isoleucine and histidine among anaerobic athletes (ORadj 2.9; 95% CI:
Citation: Baranauskas, M.; 1.1–4.7 and ORadj 2.9; 95% CI: 1.0–4.3, respectively). An inverse feasible association was indicated
Kupčiūnaitė, I.; Stukas, R. Dietary between a higher intake of valine and lower muscle mass quantities among anaerobic male athletes
Intake of Protein and Essential (ORadj 0.1; 95% CI: 0.1–0.5). The recommendations for sports nutritionists should emphasize the
Amino Acids for Sustainable Muscle
necessity of advising professional athletes on dietary strategies on how to manipulate dietary amino
Development in Elite Male Athletes.
acid composition with respect to achieving long-term body composition goals.
Nutrients 2023, 15, 4003. https://
2 doi.org/10.3390/nu15184003
Keywords: nutrition; diet; elite athletes; anaerobic sports; aerobic sports; body composition; muscle
Academic Editors: David C. Nieman development; protein; essential amino acids
and Philip J. Atherton

Received: 24 August 2023

Revised: 8 September 2023
Accepted: 14 September 2023
1. Introduction
Published: 15 September 2023 Nutrition is characterized as a certain behavior by which nutrients are consumed
in sufficient amounts for ensuring a good standard of living and maintaining a healthy
lifestyle. The daily nutritional requirements for athletes are particularly strict. More
specifically, athletes must consume nutrients in increased and balanced quantities. In
Copyright: © 2023 by the authors. addition, the nutritional profile of athletes is important for optimizing athletic performance
Licensee MDPI, Basel, Switzerland.
and depends on factors, namely sex, age, branch of sports, and athletic goals, as they appear
This article is an open access article
to be related to body composition [1]. Athletes need to develop a relatively high muscle
distributed under the terms and
mass and low body adipose tissue for the sake of better athletic performance. The striated
conditions of the Creative Commons
muscle tissue is the most metabolically active bodily tissue. Muscle proteins are unstable,
Attribution (CC BY) license (https://
as they have a permanent ability to turn over, i.e., degrade and synthesize. The changes
creativecommons.org/licenses/by/
4.0/).
in both the synthesis and degradation of muscle proteins play an important role in the

Nutrients 2023, 15, 4003. https://doi.org/10.3390/nu15184003 https://www.mdpi.com/journal/nutrients

Nutrients 2023, 15, 4003 2 of 18

recovery and remodeling of muscle proteins following physical loading. The changes in
muscle mass depend on the changes in the protein content in myofibril, accordingly. Thus,
such metabolic regulation has a key role in the adaptations of skeletal muscle (in terms of
size) to exercise training. Approximately 5% of essential amino acids (EAAs) from protein
catabolism are oxidized in muscles at partial levels and inaccessible for a new protein
translation. In all, 25% of EAAs released into the blood are used by other body tissues.
The remaining amount (70%) of essential amino acids is recycled into protein synthesis [2].
Thus, protein degradation rates always surpass the biological process of the synthesis of
new protein cells by 30% on average, depending on an insufficient EAA intake.
The higher rate of protein synthesis can only occur with protein intake in appropriate
doses [3]. It has been found that, due to aminoacidemia, the positive muscle protein net
balance can be ensured between 1.5 and 3 h after consumption of a high-protein meal
within 30 to 45 min during the postprandial state [4]. In addition, the amino acid-mediated
anabolic stimulation of muscle protein synthesis relies on the amount of dietary protein
and amino acid intake, i.e., the biological value of proteins. There are a total of 20 amino
acids that make up muscle proteins. Nine out of these 20 amino acids belong to the group
of EAAs. EAAs cannot be produced by the human body in physiologically significant
quantities, and, therefore, are necessarily obtained through an adequate diet. The remaining
11 amino acids are categorized as non-EEAs, as they might be produced by the physical
body [5,6]. Thus, the full range of nine EAAs and the 11 non-essential amino acids should
be available in appropriate amounts for protein biosynthesis [7]. Hence, the creation of new
proteins may be restricted by a limited presence of any EAA, whereas the lack of non-EAAs
could be offset by the boosted de novo synthesis [8].
On the other hand, an essential variable that can be related to the general efficacy
of protein/amino acids in the course of exercise training is the absolute protein intake
per day. The factors associated with protein timing and the quality of protein used are
equally relevant to assessing the effects of dietary protein intake during long-lasting exer-
cise participation [9]. Therefore, a large number of meta-analyses and experimental and
observational studies without intervention have been conducted to summarize the effects of
the consumption of protein supplements on the occurrence of changes in body composition,
muscular power and strength or the levels of bodily adaptation to exercise [10–20].
However, the research has focused on protein supplementation in the physically active
adult population, whereas the diet quality, nutrient intake, and amino acid composition in
the subjects’ diets were uncontrolled.
Scientific attention should also focus on the nutritional profile when athletes lack
energy/carbohydrates and protein in their diet, as both the total daily protein intake and the
quality of protein intake can be useful for the increase in and growth of muscle cells during
exercise training. This scientific theory is supported by meta-analysis, which explains
that the total protein consumption, with the modern Western diet, is a more important
factor than the daily distribution of protein intake in triggering muscle hypertrophy during
physical training [15]. Therefore, further determination of the nutritional status and body
composition in athletes remains necessary, as no specific agreement has been reached on
the results of protein intake to boost muscle hypertrophy. Additionally, so far there have
been no studies aiming to summarize the association between the muscle mass (in terms
of size) of elite athletes and the nutrient intake in most countries of the European Union
and the Baltic States. The aim of this study was to assess the association between the diet
quality profile and the training-induced muscle mass estimated by bioelectrical impedance
among elite male athletes.

2. Materials and Methods

2.1. Data Collection and Study Participants
A total of 336 Lithuanian elite athletes of the eligible population were selected from the
list approved by the Lithuanian National Olympic Committee (LNOC). Stratified random
sampling was used to enroll elite male athletes for the observational study. The following
 2. Materials and Methods
2.1. Data Collection and Study Participants

Nutrients 2023, 15, 4003

A total of 336 Lithuanian elite athletes of the eligible population were selected 3 of 18
from
the list approved by the Lithuanian National Olympic Committee (LNOC). Stratified ran-
dom sampling was used to enroll elite male athletes for the observational study. The fol-
lowing requirements
requirements were
were the mainthecriteria
mainfor criteria for of
inclusion inclusion
athletes inofthe
athletes in the
study: (1) study: (1) pro-
professional
fessional male athletes
male athletes participating
participating in the preparatory
in the preparatory training period; training period;in(2)
(2) participants theparticipants
Europe in
and World
the Europe andAthletics
WorldChampionships; (3) candidates for
Athletics Championships; (3)the Olympic Team.
candidates A more
for the detailed
Olympic Team. A
morealgorithm
detailedfor the recruitment
algorithm for theprocess is presented
recruitment in Figure
process 1.
is presented in Figure 1.

Figure 1. Enrollment flowchart for elite male athletes.

Figure 1. Enrollment flowchart for elite male athletes.

Finally, during
Finally, during the periodfrom
the period from
20182018 to 2019,
to 2019, 234
234 elite elite
male maleaged
athletes athletes
18.1 ± aged 18.1 ± 3.1
3.1 years
involved in exercises for Olympic sports, namely boxing, judo, Greco-Roman
years involved in exercises for Olympic sports, namely boxing, judo, Greco-Roman wres- wrestling,
tling,taekwondo,
taekwondo, weightlifting, basketball,
weightlifting, gymnastics,
basketball, high jump,
gymnastics, rowing,
high road
jump, cycling,road
rowing, swim-cycling,
ming, skiing, biathlon, long-distance running, and modern pentathlon, were included in
swimming, skiing, biathlon, long-distance running, and modern pentathlon, were in-
the comparative cross-sectional study (Table 1).
cluded in the comparative cross-sectional study (Table 1).
Table 1. Distribution of male athletes (in percentage) engaged in different sports according to the
Table 1. Distribution
dominant of male pathway
energy-producing athletesin(in
thepercentage)
body. engaged in different sports according to the
dominant energy-producing pathway in the body.
Eligible Analyzed Eligible Analyzed
Anaerobic Sports n = Eligible
138 Analyzed
n= 104 Aerobic Sports n = 198 Eligible n = 130Analyzed
Anaerobic Sports n n =%138 n n =%104 Aerobic Sports n %n = 198n %n = 130
Boxing 15 n 10.9 % 14 n 13.5 % Rowing 37 n
18.7 %
28 n
21.5 %
Judo
Boxing 13 15 9.4 10.9 6 14 5.813.5 Road cycling
Rowing 51 25.8
37 31
18.7 23.8
28 21.5
Greco-Roman wrestling 30 21.7 29 27.9 Swimming 44 22.2 29 22.3
Judo
Taekwondo 4 13 2.9 9.4 3 6 2.9 5.8 Road cycling 19
Skiing 51
9.6 25.8
12 31
9.2 23.8
Greco-Roman wrestling
Weightlifting 8 30 5.8 21.7 6 29 5.827.9 Swimming 22
Biathlon 44
11.1 22.2
17 29
13.1 22.3
Basketball 53 38.4 39 37.5 Long-distance running 13 6.6 8 6.2
Taekwondo
Gymnastics 4
4 2.9 2.9 2 3 1.9 2.9 Modern pentathlon Skiing 12
19
6.1
9.6
5
12
3.8
9.2
Weightlifting
Disc throw, javelin throw 7 8 5.1 5.8 3 6 2.9 5.8 - Biathlon 22 11.1
- 17
- 13.1
High jump
Basketball 4 53 2.9 38.4 2 39 1.937.5 -
Long-distance running 13 -
6.6 -8 6.2
Gymnastics 4 2.9 2 1.9 Modern pentathlon 12 6.1 5 3.8
Disc throw, javelin throw 7 5.1 3 2.9 – – –
High jump 4 2.9 2 1.9 – – –
Nutrients 2023, 15, 4003 4 of 18

The Lithuanian elite male athletes engaged in different sports were classified into two
groups, namely anaerobic (75.3%, n = 104) and aerobic (65.7%, n = 130) athletes, depending
on two primary energy-producing pathways in the body [21]. All tests related to the study
were performed during the preparatory training period prior to the competitions in a cohort
of male elite athletes. The average physical load duration in study participants matched
176.9 ± 62.6 min per day. The physical activity levels of elite male athletes fully conformed
to the intensity zones approved for training plans by the Lithuanian Sports Centre (LSC)
and LNOC (Table 2).

Table 2. Training programs for elite male athletes.

Aerobic
Anaerobic Sports
Variables Sports
(n = 104)
(n = 130)
Training period Special training Special training
Training experience, years 8.2 ± 3.7 7.8 ± 3.9
Exercise per month, days 23.3 ± 3.1 23.1 ± 3.6
Duration of training, hours per month 41.9 ± 12.1 48.6 ± 17.4
Duration of training, hours per day 2.8 ± 1.1 3.1 ± 1.1
Physical activity levels were allocated for five intensity zones depending on energy-producing pathway during workouts (% 1 )
Aerobic endurance training, recovery: heart rate equaled 130 ± 10 bpm,
10–17% 17–44%
blood lactate levels were up to 2 mmol/L
Aerobic strength training: heart rate equaled 150 ± 10 bpm, blood
lactate levels were 2–4 mmol/L, and special muscular power increased 19–41% 37–70%
at the anaerobic threshold
Aerobic and anaerobic glycolytic strength training: heart rate equaled
13–36% 9–34%
170 ± 10 bpm, blood lactate levels were 4–12 mmol/L
Anaerobic glycolytic strength training: heart rate ≥ 181 bpm, blood
0–6% 0–7%
lactate levels were up to 21 mmol/L
Anaerobic phosphocreatine strength training: blood lactate levels were
0–2% 0–4%
1.5–6 mmol/L
bpm—beats per minute; 1 —time allocated for intensity areas during workouts (%).

2.2. Anthropometric Measures

The analysis of the body composition of elite athletes was conducted at the Lithuanian
Sports Medicine Centre (LSMC). A stadiometer was used for measuring the height to the
nearest ±1 cm of athletes. Due to the bioelectrical impedance analysis (BIA) conducted
as a non-invasive testing method referring to the third level of validity approach, the
outcomes of which were highly correlated to those of dual-energy X-ray absorptiometry
(DXA) [22,23], it was possible to analyze and estimate the body composition using the
electrical resistance of various tissues of the body [24,25]. In 234 male athletes, the body
composition evaluation using the BIA method via an X-scan (Kyungsan City, Republic
of Korea) device was conducted. Impedance measurements were performed by using
5 different electrical signals of 5, 50, 250, 550, and 1000 kHz. The BIA provided information
on the body composition of athletes: body weight (BW) (kg), fatty tissue mass (FM) (kg
and percentage of BW), lean body mass (LBM) (kg and percentage of BW), muscle mass
(MM) (kg and percentage of BW). The muscle and fat mass index (MFMI) of each athlete
was calculated by dividing MM (kg) by BF (kg). For BIA outcomes and the assessment of
individual body mass components (FM, MM, and MFMI), appropriate scales were used.
These norms identified for elite athletes were previously invented, published, and currently
used by the authors for the aims of this study [26].

2.3. Nutritional Assessment

The resting metabolic rate (RMR) was estimated using the Harris–Benedict equa-
tion [27]. Twenty-four-hour physical activity recalls were gathered on the same day the
athletes reported for their daily calorie consumption. The additional energy expenditure
Nutrients 2023, 15, 4003 5 of 18

during exercise (training energy expenditure (TEE)) was estimated in the light of the recom-
mendations of the American Dietetic Association, Dietitians of Canada, and the American
College of Sports Medicine [28] as well as the report of empirical research by Ainsworth
and colleagues [29].
Considering the fact that there is currently no “gold standard” for assessing di-
etary intake, dietary recalls, as the most common approach used in sports nutrition re-
search [1,30–35], were also applied in our study. The 24-h dietary recall method was used
in the dietary assessment of athletes [28,30,36,37]. The participants were instructed to
maintain their habitual diet throughout the study and finally were enrolled to complete the
forms for their 24-h dietary recalls of three non-consecutive days with the assistance of a
sports dietitian [38]. The forms with dietary recalls were collected from all participants by a
sports dietitian using a direct personal interview method during the physical examination
of athletes at the LSMC. All food and drinks consumed by the study participants were
recorded by the trained interviewer in line with the basis of the amounts of food provided
in the Atlas of Foodstuffs and Dishes [39]. A list of the athletes’ average daily food intakes
was compiled during the next data processing phase. The nutritional analysis software
NutriSurvey (the English translation of a professional German nutrition software program
(EBISpro)) was applied for tracking the ingredients used in food recipes in order to calculate
the nutrition values of individual food items (http://www.nutrisurvey.de/ (accessed on 5
January 2018)). Additionally, the NutriSurvey function "Food/Include more foods from
other databases" was used as well as the nutrition values of food items manually integrated
from the Lithuanian food database [40].
Each sport discipline is based on the origin of training and is related to one of two
primary energy-producing pathways in the body along with nutrient requirements. There-
fore, sports nutritionists working with athletes involved in different sports (anaerobic and
aerobic) have been provided with nutrition guidelines established by the international
sporting committees, namely, the International Society of Sports Nutrition (ISSN), the
International Olympic Committee (IOC), and the American College of Sports Medicine
(ACSM). Consequently, the intakes of carbohydrates and protein in the athletes we studied
were assessed in accordance with the recommended values provided in the these scientific
recommendations [41–46]. More specifically, the carbohydrate content recommended for
athletes is consistent with 7–10 g/kg/day. The protein intake for athletes should be within
the limits of 1.4 to 2.0 g/kg/day [47,48]. Taking into account that some uncertainty remains
over the adult EAA requirements, the World Health Organization (WHO) has proposed
a recalculation of the individual amino acid requirements divided by the total protein
requirement. In this context, the EAA requirements for athletes were assessed in accor-
dance with the recommendations made by the WHO, following an empirical recalculation
in accordance with the standards applicable to athletes [49,50]. Additionally, the widely
used technique for nutritional assessment was applied for estimating the nitrogen balance
(NB) in male athletes. The NB of all of the athletes was calculated using the formula: NB
(g/N/day) = DNI − UNA − NUN − UNPL, where (1) DNI—dietary nitrogen intake
estimated as dietary protein intake (DPI)/6.25 [51]; (2) UNA—urea nitrogen appearance;
(3) NUN—non-urinary nitrogen excretion (e.g., ammonia, uric acid, creatinine, amino
acids) [52,53] estimated as 31 mg/kg [54]; (4) UNPL—urinary nitrogen protein losses (esti-
mated as 2 g for gastrointestinal and integumentary (dermal) losses) [55]. The data were
rearranged to empower the estimation of UNA from protein intake (using Bergstrom’s
formula, UNA = (DPI − 19)/7.62) [56].
In the meantime, fat intake may fluctuate between 20% and 35%, contingent on the
daily caloric consumption by athletes.

2.4. Statistical Analysis

The cross-sectional study was conducted in accordance with the Strengthening the
Reporting of Observational Studies in Epidemiology (STROBE) checklist [57].
Nutrients 2023, 15, 4003 6 of 18

Statistical analysis of the empirical data of the study was executed using both Statistical
Package for the Social Sciences (SPSS) V.25 for Windows (Armonk, NY, USA) and Stata
version 12.1 (StataCorp, College Station, TX, USA). The Shapiro–Wilk W test was used
to check the normality of the data. If the data normality was confirmed, the paired two-
sample t-tests were used to assess the significance of the differences between the central
tendency measures (mean ± standard deviation (SD)) of the two categories. Cohen’s d
(d) as an effect size was also evaluated to accompany the reporting of t-test results as well
as to indicate the standardized difference between the two means. The effect sizes were
interpreted as follows: 0.2 ≤ d < 0.5 (small effect), 0.5 ≤ d < 0.8 (moderate effect), and
0.8 ≤ d (large effect). If d was larger than 1, the difference between the two means was
larger than one SD, and anything larger than 2 signified that the difference was larger than
two SDs [58]. The Pearson correlation coefficient (r), which is the most common way of
measuring a linear correlation, was used to set up the relationship between the variables
such as dietary protein intake, nitrogen balance, and muscle mass in a sample of male
athletes. The correlation coefficients were interpreted as follows: 0 ≤ r < 0.3 (negligible
correlation), 0.3 ≤ r < 0.5 (moderate correlation), and 0.5 ≤ r < 1 (high correlation). All of
the reported p-values were developed on two-sided tests and compared to the significance
level of 5%.
In addition, multiple logistic regression analyses with a binary dependent variable
among two subgroups covering the anaerobic and aerobic sports were performed. The de-
pendent variables, namely muscle mass (MM) (in kg) of anaerobic athletes
(61 kg ≤ MM > 61 kg) and MM (in kg) of aerobic athletes (57 kg ≤ MM > 57 kg), were
adjusted to the dichotomous form (1—MM was below median value in the subgroup (refer-
ence category), 2—MM was equal to or larger than the median value in the subgroup). The
units of measurement of independent variables, namely the intake of energy (kcal/kg/day),
macronutrients (g/kg/day), and EAAs (mg/g of protein/day), were adjusted to the di-
chotomous form relying on cut-off values that were determined using the median values of
the subgroups of anaerobic and aerobic male athletes. The logistic regression models were
adjusted for the age and training experience of professional male athletes. Goodness-of-fit
of logistic regression models was evaluated using the Nagelkerke R2 statistic.

3. Results
3.1. Body Composition of Elite Male Athletes
Body composition is a physical measurement that provides more specific information
about physical fitness than BW alone. Body composition can be defined as the proportion
of fat and muscle mass (MM) in the body. The body composition of Lithuanian elite male
athletes was assessed as indicated in Table 3. LBM and MM in male athletes fluctuated
within the normal range. Despite the sporting discipline, it was also found that the
measures of central tendency for LBM were significantly higher than the mean norm values
in both anaerobic and aerobic athletes. The ∆ LBMin% (actual LBMin% —avg. recommended
LBMin% ) values for anaerobic and aerobic athletes were 3.3% (95% PI: 2.2; 4.3, d = 0.6) and
3.3% (95% PI: 2.6; 4.1, d = 0.8), respectively. The mean BF values for male athletes involved
in both anaerobic and aerobic sports (16.7 ± 5.2% and 16.7 ± 4.2%) exceeded the levels of
optimum values (10–14%) by 4.7% and indicated only acceptable body fat levels (d = 0.2
and d = 0.2, respectively). However, irrespective of relatively higher study participant
BF levels, MFMI was extensive in male athletes. The t-tests between MFMIs in anaerobic
and aerobic male athletes (5.3 ± 2.4 and 5.2 ± 2.6, respectively) and the avg. extensive
MFMI limit (4.7–6.0) were found to be without statistical significance: ∆ MFMIs (actual
MFMI—avg. extensive MFMI) of anaerobic and aerobic athletes were −0.1 (95% PI: −0.5;
0.4, d = 0.02) and −0.2 (95% PI: −0.6; 0.3, d = 0.05), respectively.
Nutrients 2023, 15, 4003 7 of 18

Table 3. Body composition of male athletes depending on sports.

Anaerobic Aerobic
Normative
Variables Sports Sports ∆ana (95% CI) D ∆aer (95% CI) d
Data
(n = 104) (n = 130)
Height (m) 1.8 ± 0.2 1.8 ± 0.1 - - - - -
Body weight (kg) 77.5 ± 17.4 75.1 ± 11.6 - - - - -
Lean body mass (kg) 63.8 ± 11.4 62.2 ± 7.7 - - - - -
Lean body mass (% of BW)BIA 83.2 ± 5.3 83.3 ± 4.2 75–85 b (80 a ) 3.3 (2.2; 4.3) 0.6 3.3 (2.6; 4.1) 0.8
Muscle mass (kg) 59.3 ± 10.5 57.9 ± 7.0 - - - - -
Muscle mass (% of BW)BIA 77.4 ± 5.2 77.6 ± 4.1 74–80 c (77 a ) 0.4 (−0.6; 1.4) 0.1 0.6 (−0.1; 1.3) 0.1
Body fat (kg) 13.7 ± 7.1 12.9 ± 4.7 - - - - -
Body fat (% of BW)BIA 16.7 ± 5.2 16.7 ± 4.2 10–14 d (12 a ) 4.7 (3.7; 5.7) 0.2 4.7 (3.9; 5.4) 0.2
Muscle and fat mass index 5.3 ± 2.4 5.2 ± 2.6 4.7–6.0 e (5.4 a ) –0.1 (−0.5; 0.4) 0.02 −0.2 (−0.6; 0.3) 0.05
Data are presented as means ± SD; SD—standard deviation; BW—body weight; ∆—delta; ∆ana —actual measure
of anaerobic athletes–recommended levels for anthropometric measurements; ∆aer —actual measure of aerobic
athletes–recommended levels for anthropometric measurements; 95% CI—95% confidence intervals; d—effect size
(d); a —measure of central tendency; b —lean body mass norm for male is 75–85%; c —muscle mass norm for male
is 74–80%; d —optimal body fat for male athletes is 10–14% [26]; e —extensive muscle and fat mass index for male
athletes is 4.7–6.0 [26].

3.2. Nutritional Profile of Elite Male Athletes

Table 4 shows the reported usual mean intake of energy, carbohydrates, fats, proteins,
and amino acids of the elite male athletes. Regardless of sport, the mean fat energy of
40 ± 7.6% found in the diets of male athletes exceeded 35% of calories. The carbohydrate-
deficient diet (5.7 ± 0.6 g/kg/day) was usual only among aerobic athletes: ∆ carbohydrates
actual intake—avg. recommended) of aerobic athletes was −2.9 (95% PI: −3.2; −2.5, d = 4.7).
Meanwhile, the mean protein intake of 1.8 ± 0.6 g/kg/day was within the recommended
limits of 1.4–2.0 g/kg/day in both anaerobic and aerobic male athletes. Consequently,
in terms of effect size (d), it can be affirmed that the reported intakes of EAAs such as
leucine, isoleucine, valine, phenylalanine plus tyrosine, methionine, lysine, tryptophan,
threonine, and histidine were within plus 2 standard deviations around mean requirements
and revealed over-consumption of all EAAs in the population of elite male athletes [45];
thus, these rates could be assigned to large effect sizes (2.6 ≤ d ≤ 6.3).
Furthermore, if an amino acid such as leucine acts as a stimulating precursor for
muscle protein synthesis and has higher upper limits for recommended daily allowances
(up to 12 g/day) [47,48,50], the means of leucine intake for anaerobic and aerobic athletes
were calculated as follows: 10.2 ± 4.5 g/day and 10 ± 3.3 g/day, respectively.
Also, our study confirmed that the mean 24-h nitrogen balance of 2.0 ± 1.3 g in athletes
was positive and related to a sufficient protein intake or consumption of more protein than
the body needs, and was also optimal for anabolism.

3.3. Association between Protein Intake and Muscle Mass

Figure 2 shows the relationship between the nitrogen balance (g/day) and the size
of muscle mass (kg) in elite male athletes. Taking into consideration that the current
study indicated a net 24-h positive nitrogen balance of 2 g in both anaerobic and aerobic
male athletes, it can be summarized that a positive nitrogen balance during the periods
of adequate nutrition was correlated with the potential promotion of muscle growth only
in the cases where anaerobic training acted as a stimulus for nitrogen/protein retention
(r = 0.23, p = 0.009).
The results of the multivariate analysis are displayed in Figure 3. Two multivariate
logistic regression models were constructed to understand how the determinants related to
dietary intake may predict muscle mass in the samples of anaerobic and aerobic elite male
athletes. Multivariate logistic regression models were adjusted for the age and training
experience of male athletes.
 balance; ∆aer—actual nutrient intake and nitrogen balance in aerobic athletes—recommended leve
for nutrient intake and nitrogen balance; 95% CI—95% confidence intervals; d—effect size (d); a—
Nutrients 2023, 15, 4003 measure of central tendency; 1—essential amino acid (EAA) (mg/g of protein) intake 8 ofin
18anaerob
athletes; —EAA (mg/g of protein) intake in aerobic athletes; —EAA requirements (mg/g of protein
2 3

EAA requirements: the best current estimates have been reported by the World Health Organizatio
(WHO)
Table [49]. EAA requirements
4. Nutritional for athletes
profile and nitrogen balancehave been
of male assessed
athletes in accordance
according to sports. with the recommen
dations made by the WHO, following an empirical recalculation in accordance with the standard
Anaerobic Aerobic
Nutrient Intake and applicable to athletes [47,48,50]; Leu—leucine; Iso—isoleucine; Val—valine; Phe + Tyr—phenylal
Sports Sports Requirements ∆ana (95% CI) d ∆aer (95% CI) d
Nitrogen Balance nine plus tyrosine; Met—methionine; Lys—lysine; Try—tryptophan; Thr—threonine; His—hist
(n = 104) (n = 130)
Energy intake, dine.
50 ± 15.9 49.6 ± 12.8 55.6 ± 4.2 −6.3 (−8; −4.6) 0.5 −6.2 (−8.2; −4.2) 0.6
kcal/kg/day
Fat, % 39.6 ± 6.9 40.3 ± 8.2 25–35 (30 a) 10.3 (8.8; 11.7) 1.4 9.6 (8.3; 10.9) 1.3
Carbohydrates, g/kg/day 5.6 ±Furthermore,
1.9 5.7 ± 0.6if an amino
7–10 (8.5 acid
a) such
−2.9 (−as3.5;leucine
−3.3) acts
1.5 as a −
stimulating
2.9 (−3.2; −2.5) precursor
4.7 for mus
± 0.7 1.8 ± 0.6 a) 0.1 (−0.1; 0.2)
Protein, g/kg/day cle1.8 protein synthesis and has
1.4–2.0 higher
(1.7 upper
0.1 (–0.1; limits
0.2) for0.1recommended daily allowances
0.1 (u
Leu, mg/g of protein/day 76 ± 6.6 1 76 ± 5.8 2 59 3 16.8 (15.5; 18) 2.6 17 (15.9; 18) 2.9
Iso, mg/g of protein/day to4512 g/day)
± 3.2 1 [47,48,50],
45 ± 3.4 2 the means
30 3 of leucine
14.5 (13.8;intake
15) for4.7anaerobic and
14.7 (14; aerobic4.4athletes wer
15.3)
Val, mg/g of protein/day ± 1 ± 2 3 14.1 (13.2; 15) 3.3 13.7 (13; 14.4) 3.3
calculated as follows: 10.2 ± 4.5 g/day and 10 ± 3.3 g/day, respectively.
53 4.3 53 3.8 39
Phe + Tyr, mg/g of 1 2 3
80 ± 6.8
Also, 79 ±study
our 6.5 38
confirmed 41.7 (40.4;
that the mean 43) 24-h nitrogen
6.2 41.5 (40; 42.6) of 2.0 6.3
balance ± 1.3 g in ath
protein/day
Met, mg/g of protein/day 22 ± 2.2 1 22 ± 2.2 2 16 3 5.9 (5.5; 6.3) 2.7 6.2 (5.8; 6.5) 2.7
letes was positive 2and related to a sufficient protein intake or consumption of more pro
Lys, mg/g of protein/day 67 ± 5.9 1 67 ± 6.2 45 3 21.8 (21; 22.9) 3.7 21.7 (20.7; 23) 3.5
Try, mg/g of protein/day tein
13 ± than
1.5 1 the13body
± 1.4 2needs, and
6 3 was also 7.1optimal
(6.8; 7.4) for anabolism.
4.7 7.4 (7.2; 7.7) 5
Thr, mg/g of protein/day 39 ± 2.8 1 39 ± 2.7 2 23 3 16 (15; 16.6) 5.7 16.4 (16; 16.9) 5.9
His, mg/g of protein/day 28 ± 3.2 1 28 ± 2.9 2 15 3 13.2 (12.7; 14) 4.1 13.4 (13; 13.9) 4.5
24 h nitrogen balance, g 3.3. Association
2.0 ± 1.6 between
1.9 ± 1.1 Protein
≥1 Intake and Muscle
0.9 (0.7; 1.1) Mass0.6 9.6 (8.3; 10.9) 0.8
Figure
Data 2 shows
are presented the±relationship
as means deviation; ∆—delta;
SD; SD—standardbetween ∆ana —actual
the nitrogen balance
nutrient (g/day) and the size o
intake and nitrogen
balance in anaerobic athletes—recommended levels for nutrient intake and nitrogen balance; ∆aer —actual nutrient
muscle mass (kg) in elite male athletes. Taking into consideration that the
intake and nitrogen balance in aerobic athletes–recommended levels for nutrient intake and nitrogen balance; current stud
95% CI—95% confidence intervals; d—effect size (d); a —measure of central tendency; 1 —essential amino acid
indicated a net 24-h positive nitrogen balance of 2 g in both anaerobic and aerobic mal
(EAA) (mg/g of protein) intake in anaerobic athletes; 2 —EAA (mg/g of protein) intake in aerobic athletes;
athletes,
3 —EAA it can be summarized
requirements (mg/g of protein).thatEAAarequirements:
positive nitrogen balance
the best current during
estimates the
have been periods
reported by of ade
the World
quate Health Organization
nutrition was (WHO) with
correlated [49]. EAA
the requirements
potential for athletes have been
promotion of assessed growth
muscle in accordance
only in th
with the recommendations made by the WHO, following an empirical recalculation in accordance with the
cases where
standards anaerobic
applicable training
to athletes [47,48,50];acted as a stimulus
Leu—leucine; forVal—valine;
Iso—isoleucine; nitrogen/protein retention (r = 0.23
Phe + Tyr—phenylalanine
plus tyrosine; Met—methionine; Lys—lysine; Try—tryptophan; Thr—threonine; His—histidine.
p = 0.009).

Figure 2. (A) The relationship between the NB (g/day) and the size of muscle mass (kg) in anaerobic
Figure 2. (A) The relationship between the NB (g/day) and the size of muscle mass (kg) in anaerob
male athletes (r = 0.23, p = 0.009); (B) the relationship between the NB (g/day) and the size of muscle
male athletes
mass (kg) in(r = 0.23,male
aerobic p = athletes
0.009); (r
(B)= the
0.12,relationship
p = 0.1). between the NB (g/day) and the size of musc
mass (kg) in aerobic male athletes (r = 0.12, p = 0.1).
The ORsadj for increased muscle mass were associated with higher intakes of protein
(OR adj 2.2;
The 95% CI:
results 1.3; 3.7)
of the as well as EAAs
multivariate suchare
analysis as isoleucine
displayed (OR 2.9; 95%
inadjFigure 3.CI:
Two1.1;multivariat
4.7)
and histidine
logistic regression(ORadj 2.9; 95%
models CI: 1.0;
were 4.3) in a group
constructed of anaerobic how
to understand athletes.
theIndeterminants
addition, an relate
inverse association (ORadj 0.1; 95% CI: 0.1; 0.5) was observed between higher intakes of
to dietary intake may predict muscle mass in the samples of anaerobic and aerobic elit
valine and muscle development in anaerobic athletes.
male athletes. Multivariate
In contrast, there was nologistic regression
relationship betweenmodels wereintake
the increased adjusted for the
of protein or age
EAAsand train
ingand
experience
the size of ofmuscle
male athletes.
mass in a sample of aerobic athletes. The results of this study
revealed only the association (ORadj 0.3; 95% CI: 0.1; 0.7) between the lower-carbohydrate
diet and the decrease in muscle mass among well-trained and elite endurance male athletes.
Nutrients 2023, 15, 4003 9 of 18
Nutrients 2023, 15, 4003 9 of 18

Figure
Figure 3. The
3. The associationbetween
association between the
the intake
intakeofof
macronutrients
macronutrients and and
essential amino amino
essential acids (EAAs)
acids (EAAs)
and muscle mass (in kg) as a dependent variable in the samples of anaerobic
and muscle mass (in kg) as a dependent variable in the samples of anaerobic and aerobic and aerobic malemale ath-
athletes (multivariate analyses). OR —adjusted odds ratio (OR = e β ); 95% CI—95% confi-
letes (multivariate analyses). ORadj—adjusted odds ratio (ORadj = e ); 95% CI—95% confidence inter-
adj adjβ
dence interval; *—p-value 1 —reference category: muscle mass ≤ 61 kg; 1 —Nagelkerke
< 0.05. category:
val; *—p-value
2
< 0.05. 1—reference muscle mass ≤ 61 kg; 1—Nagelkerke R2 = 0.59; the lo-
R = 0.59; the logistic regression model was adjusted for the age and training experience of anaer-
gistic regression model2 was adjusted for the age and training experience of anaerobic male athletes.
obic male athletes. —reference category:
2—reference muscle mass ≤ 57; 2 —Nagelkerke R2 = 0.29; the lo-
category: muscle mass ≤ 57; 2—Nagelkerke R2 = 0.29; the logistic regression model was
gistic regression model was adjusted for the age and training experience of aerobic male ath-
adjusted for the age and training experience of aerobic male athletes. BW—body weight; EI—energy
letes. BW—body weight; EI—energy intake; CHO—carbohydrates; PRO—protein; Leu—leucine;
intake; CHO—carbohydrates; PRO—protein; Leu—leucine; Iso—isoleucine; Val—valine; Phe +
Iso—isoleucine; Val—valine; Phe + Tyr—phenylalanine plus tyrosine; Met—methionine; Lys—lysine;
Tyr—phenylalanine plus tyrosine; Met—methionine; Lys—lysine; Try—tryptophan; Thr—threo-
Try—tryptophan; Thr—threonine; His—histidine.
nine; His—histidine.
4. Discussion
4.1.
TheAthletic
ORsadjBody
for Composition
increased muscle mass were associated with higher intakes of protein
(ORadj 2.2; 95%body
When CI: 1.3; 3.7) as well
composition as EAAs
was one such astriggering
of the factors isoleucine (ORadj 2.9; 95%
exercise-induced CI: 1.1; 4.7)
adapta-
tions, our study findings revealed both the high development of the musculoskeletal
and histidine (ORadj 2.9; 95% CI: 1.0; 4.3) in a group of anaerobic athletes. In addition, an mass
and the significant increase in the body fat mass percentage in both groups of Lithuanian
inverse association (ORadj 0.1; 95% CI: 0.1; 0.5) was observed between higher intakes of
male elite athletes. We compared our study’s empirical data with those described by
valine and muscle development in anaerobic athletes.
other reports on the body composition assessment of professional male athletes through
In contrast, therepublished
bioimpedanciometry was no in relationship
Canada [59], between
the United the increased
States of Americaintake of protein or
(USA) [60,61],
EAAs and the
Portugal size
[62], of muscle
Australia mass [64,65],
[63], Spain in a sample
Turkeyof[66],
aerobic
Russiaathletes. The results
[67,68], Taiwan of this study
[69], Indone-
sia [70],
revealed Republic
only of Korea [71],
the association Poland
(OR adj 0.3;[72],
95%and
CI:Czech [73].between
0.1; 0.7) The cohorts
theoflower-carbohydrate
professional
male athletes from the target countries provided the mean
diet and the decrease in muscle mass among well-trained and elite enduranceoutcomes for LBM (%), BF
male ath-
(%), and MFMI as follows: 84.2%, 14.9%, and 5.8 for male athletes engaged in anaerobic
letes.
sports such as volleyball [59], football [60], judo [62,66], boxing [63], track and field [64], ski
jumping [67], wrestling [69], and weightlifting [70], and 84.3%, 14.9%, and 5.2 for male ath-
4. Discussion
letes from aerobic sports, namely rowing [65], road cycling [61], cross-country skiing [71],
biathlon [69],
4.1. Athletic Bodylong-distance
Compositionrunning [72], and pentathlon [73]. It should be highlighted that
the mean MFMIs of the elite anaerobic male athletes we studied were lower than the MFMI
Whenofbody
values composition
well-trained sportsmenwasfrom
oneforeign
of thecountries
factors triggering
(5.2 (95%CI: exercise-induced
4.8; 5.8) vs. 5.8 (95%adapta-
tions, our study findings revealed both the high development of
CI: 3.9; 7.6)). Thus, our study finding regarding the poorer body compositionthe musculoskeletal
status can be mass
and explained
the significant
only byincrease
the higherinfatthe body
mass fat mass
percentage percentage
(~17%) found in in both groups
Lithuanian of Lithuanian
male athletes
compared to the figures for the average fat levels of high-performance anaerobic
male elite athletes. We compared our study’s empirical data with those described by other athletes
fromon
reports other
thecountries (~15%).
body composition assessment of professional male athletes through bioim-
pedanciometry published in Canada [59], the United States of America (USA) [60,61], Por-
tugal [62], Australia [63], Spain [64,65], Turkey [66], Russia [67,68], Taiwan [69], Indonesia
[70], Republic of Korea [71], Poland [72], and Czech [73]. The cohorts of professional male
Nutrients 2023, 15, 4003 10 of 18

4.2. Nutritional Status of Athletes

The essential nutrients in the diets of studied Lithuanian elite male athletes were
unbalanced due to an increased fat intake. A carbohydrate-deficient diet was found
only among the athletes training for endurance-based sports. The study findings on
low carbohydrate intakes of male athletes were consistent with the results obtained by
other scientists who also revealed low carbohydrate consumption, which was felt to be
significantly short of suggested levels for aerobic athletes [74–77].
An insufficient intake of carbohydrates by athletes can lead to the deterioration of
aerobic capacity, may cause rapid fatigue during exercise, can lead to exercise-related
immunodepression, and increase the risk of overtraining. As a result, muscle adaptations to
aerobic training may be slowed down [78], coupled with performance impairments [79–83].
Therefore, this lack of a nutritional profile related to carbohydrate deficit among endurance
male athletes should be corrected appropriately by converting a high-fat diet to a high-
carbohydrate diet.
Also, it should be mentioned that both aerobic and anaerobic athletes adopted a diet
high in protein. According to our study, the average amount of protein consumed by elite
male athletes amounted to 1.8 g/kg/day and fully complied with the current international
Recommended Dietary Allowance (RDA) for protein. Both the International Olympic
Committee (IOC) and the International Society of Sports Nutrition (ISSN) have reported
that, in order to maintain a positive nitrogen balance, professional athletes may benefit from
approximately twice the RDA of protein (1.4–2.0 g/kg/day) [47,48]. A similar situation
was observed among foreign professional athletes who had adopted a diet high in protein
and adequately met recommendations for protein intake [1,31,32,59,84–92].
In addition, it is recommended that elite athletes should consume all EAAs with food
in order to ensure the whole-body protein synthesis and to achieve maximum muscle mass
gain. Our study showed that there were no differences in amino acid consumption in
the samples of aerobic and anaerobic athletes. It should be highlighted that the uptake
of amino acids for protein remodeling after the absorption process may vary due to the
impact of the type of sport. Amino acids from extracellular space are used for muscle
protein synthesis following resistance exercises. In contrast, new mitochondrial protein
synthesis is maintained by the utilization of amino acids from intracellular space. Thus,
there is no difference between the recommendations for resistance and endurance athletes
in terms of the intake of protein/amino acids.
Furthermore, our analysis of the qualitative composition of amino acids consumed
by anaerobic male athletes showed that the dietary intakes of all EAAs were significantly
higher than the recommended ones. Moreover, this study focused on the intake of amino
acid, namely leucine, as an essential stimulant of muscle protein synthesis. Although
eating a diet of high-quality essential amino acid-rich protein containing 0.7 to 3.0 g
(2–12 g/day) of leucine in evenly divided doses over the day (a schedule of every 3–4 h)
is recommended by the IOC and ISSN [47,48,50]; hence, our study documented relatively
high levels (10 g/day) of leucine intake in both anaerobic and aerobic sportsmen. The
diets higher in leucine quantities (~10–14 g/day), although without significant correlation
with the magnitude of muscle mass, were identified in our previous study dealing with
the nutritional assessment of male athletes engaged in sports such as rowing, including
canoeing and kayaking, too [93].

4.3. Nutritional Intake and Muscle Mass

4.3.1. Carbohydrates for Muscle Development
This study found an association between the decreased size of muscle mass and
the reduced intake of carbohydrates in a sample of aerobic athletes. The findings of the
present study, related to the risk of a low-carbohydrate diet leading to a potentially nega-
tive effect on the synthesis of new myofibrillar proteins, can be explained by the fact that
the ingestion of carbohydrates and amino acids immediately after exercise promotes the
glucose-stimulated hormone insulin secretion and may theoretically inhibit the rate of
Nutrients 2023, 15, 4003 11 of 18

protein degradation. However, a combined intake of carbohydrates and amino acids after
exercise appears not to be any more a trigger for myofibrillar protein synthesis than the
consumption of an amino acid mixture in an isolated form. Therefore, with the purpose of
achieving training-induced muscle growth, only food high in protein/amino acids (with or
without carbohydrates) for consumption is sufficient after exercise [94,95]. On the other
hand, when a diet lacks carbohydrates, aerobic athletes exercise with decreased glycogen
stores in their muscles and liver, a condition that can alter amino acid oxidative losses dur-
ing exercise [96]. Thus, additional questions on protein requirements for endurance athletes
that take into account the impact of carbohydrate availability still need to be addressed.

4.3.2. Protein/EAAs for Muscle Development

This study demonstrated the association between a higher intake of protein and
possible anaerobic training-induced muscle gain.
The scientific literature has well documented that the branched-chain amino acids
(BCAAs) such as leucine, valine, and isoleucine correspond to almost 50% of muscle protein
EAAs [5,6]. More specifically, leucine alone is capable of initiating the process of protein
synthesis by triggering activation of the skeletal muscle mammalian target of rapamycin
complex 1 (mTORC1), the protein complex that controls protein synthesis. After initiating
this nutrient-sensing pathway, all amino acids are used in the synthesis of new muscle
proteins [97]. However, the results of leucine intake (~10 g/day) in the male athletes we
studied did not correlate with the scientific data on the outcomes of BCAA/leucine on
muscle protein synthesis and possible muscle mass gain.
There are some explanations for these inconsistent findings. Firstly, as Santos et al. [98]
speculated on the necessity of consumption of all EAAs to achieve optimal post-exercise
muscle protein remodeling, supplementing a diet with isolated forms of BCAAs was no
longer recommended for athletes [98]. In the second place, when consumed together, after
absorption into the bloodstream, the BCAAs and other amino acids compete with each
other for transport into the intestinal cells and myocytes. Therefore, single amino acid
overconsumption may lead to the limitation of protein synthesis activation as a result of
the decreased entrance of other EAAs into the muscle cells [99,100]. This type of pathway
was assigned to explain the association between the increased muscle mass and higher
intakes of isoleucine and histidine as well as to interpret our opposite study data concerning
an inverse association between a higher valine intake and the decreased level of muscle
hypertrophy in anaerobic male athletes. These findings can be explained by the likelihood
that BCAAs such as valine and leucine were unbalanced in athletes’ diets due to higher
intakes of isoleucine and histidine. More specifically, some documented evidence indicates
that all of the BCAAs and other amino acids (histidine, tryptophan, tyrosine) are actively
transferred into the human body cells via a neutral and identical heterodimeric membrane
transport protein (LAT1) [101]. Thus, in this case, the consumption levels of both leucine
and valine may not be balanced for benefiting the myofibrillar hypertrophy in the male
athletes we studied.
However, histidine, unlike valine and leucine, has been shown to have a potential
role in muscle hypertrophy in the anaerobic athletes we studied. This finding can be
attributed to the fact that histidine is a precursor of dipeptide carnosine, which is composed
of the two amino acids beta-alanine and histidine. Carnosine is an intracellular buffer
that protects skeletal muscle cells from the damaging effects of acids (lactate and other
acidic by-products of anaerobic metabolism). Consequently, the supplementation of the
diets of athletes with histidine and/or beta-alanine may be an effective ergogenic aid for
exercise lasting 1–4 min [101]. Attention must also be devoted to the evidence that due to a
decrease in intracellular pH during exercise, the amino acid transmembrane transporter
activity is also reduced, which can lead to reduced amino acid uptake into muscle cells
while reducing the muscle protein synthesis rate following exercise [99].
Nutrients 2023, 15, 4003 12 of 18

4.4. Limitations
This comparative cross-sectional study coincided with several limitations. First, a
limitation of this study was associated with the fact that the findings referring to the
relationship between the nutritional profile and muscle mass quantities among professional
athletes did not allow for causal conclusions. Therefore, cohort studies in design together
with randomized controlled trials (RTCs) are warranted to identify the effect of the optimal
dietary amino acid ratio on body composition, especially in athletic populations.
Second, our study findings are only applicable to elite male athletes and might not
be generalizable to other populations. Unfortunately, there is a lack of research on how to
optimize nutrition for females in terms of the disparities in health and physiology. In terms
of female-specific physiology for athletes, the findings of male athletes may be misapplied
to female athletes [102,103]. It was “more difficult” to recruit females for the study due
to higher hormonal intricacy [104], possible risk of low energy availability [105], and the
female athlete triad [106]; therefore, sportswomen were not included in our research. Future
research must focus on a cohort of high-power and high-endurance female athletes with
distinct body composition characteristics and metabolic profiles [107].
Third, it has been widely documented that self-reported dietary intakes can result in
biases due to underestimated outcomes, as observed in a number of cohorts, including
adolescents [108], overweight persons [109], and athletes [110–112]. Also, despite the food
records approach being considered a "gold standard" in nutritional status assessment, it cre-
ates difficulties for participants to document information correctly and accurately, as well as
to trust that researchers will correctly encode the data using the relevant databases [113–115].
Along these lines, three non-consecutive day diet logs derived from single administrations
of 24-hour dietary records were incapable of accounting for day-to-day variation and
potentially contributed to trivial biases of empirical data interpretation.
Fourth, although the DXA preferred as a "gold standard" approach leads only to
an average accuracy in estimating body fat levels, and there is no gold-standard body
composition analysis technique [84–122], the bioelectrical impedance analysis (BIA) method
referring to a third level of validity has been constructed [22]. Furthermore, scientific
evidence refers to very strong correlations between the DXA and different BIA models [23].
Thus, in our case, the body composition assessments were limited to BIA. Conclusively,
the application of the DXA method with higher levels of consistency and lower biases is
needed to match our outcomes with the male athlete body composition results reported by
other scientific researchers [123].
Finally, although our study focused on the adjusted odds ratios of dietary macronutri-
ents/EAAs for musculoskeletal mass, muscle development is multivariable and may be
determined by more potential factors such as the specificity of sports disciplines, training
phases and periods within a macrocycle, intensities used during training loads, exercise-
induced adaptation levels, etc.

5. Conclusions
All elite male athletes showed the consumption of lipids above the recommended
levels. The present study highlighted a significant macronutrient imbalance due to de-
creased carbohydrate intake in a group of aerobic male athletes. Notably, research by
Syed-Abdul et al. [124] explored the effects of self-implemented carbohydrate cycling and
resistance exercise on body fat reduction in anaerobic athletes, shedding light on the
connection between dietary strategies and body composition changes.
The mean daily protein intake of 1.8 g/kg/day was optimal for anabolism in the
samples of both anaerobic and aerobic male athletes. Nevertheless, protein intake in
appropriate doses was associated with a potential muscle mass gain only in anaerobic male
athletes. Regardless of sufficient levels of anabolism-inducing protein consumption, our
study revealed the relationship between a carbohydrate-deficient diet and the decrease in
muscle mass among aerobic male athletes.
Nutrients 2023, 15, 4003 13 of 18

Since sufficient availability of all amino acid precursors is a prerequisite for increasing
the rate of protein synthesis in muscles, the findings of this study disclosed the positive
association between the possible muscle mass gain and the increased intakes of amino
acids such as isoleucine and histidine among anaerobic athletes. Our study also revealed
an inverse relationship between a higher intake of valine and a decreased level of feasible
muscle hypertrophy in anaerobic athletes. Evidently, in terms of the quality of dietary
amino acid composition, the increased intake of essential amino acids such as isoleucine
and histidine may potentially contribute to imbalances in diet quality, and this type of
imbalance may determine the intramuscular availability of other essential amino acids
following the absorption process.
Finally, this study highlighted that dietary amino acid composition is as important
as the total protein intake. Therefore, it is recommended that sports dieticians organize
meal plans for athletes that are not only adjusted to the total protein content, but optimized
for dietary amino acid composition, too. More importantly, taking into consideration
the generalized data of this study, the recommendations for sports nutritionists should
emphasize the necessity of nutrition assessment and recommendations for sportsmen
regarding their dietary strategies on how to manipulate dietary amino acids composition
with respect to achieving long-term body composition goals. However, alterations in
muscle cells may not lead to the whole-body response to physical loads. Consequently,
the benefits of dietary amino acid balance for muscle development along with physical
adaptation should be considered only as probabilistic.

Author Contributions: Conceptualization, M.B. and R.S.; methodology, M.B.; software, M.B.; valida-
tion, M.B. and R.S.; formal analysis, M.B.; investigation, M.B.; resources, M.B. and I.K.; data curation,
M.B. and R.S.; writing—original draft preparation, M.B.; writing—review and editing, R.S., I.K. and
M.B.; visualization, M.B.; supervision, R.S; project administration, I.K. and R.S. All authors have read
and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The study was conducted in accordance with the Declaration
of Helsinki, and approved by the Vilnius Regional Committee of Biomedical Research Ethics (protocol
code 158200-17-898-419, of 11 April 2017).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Data are available on request.
Acknowledgments: To the Lithuanian Sports Medicine Centre and the Lithuanian Sports Centre for
their technical support in testing the body composition of elite athletes.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Jenner, S.L.; Buckley, G.L.; Belski, R.; Devlin, B.L.; Forsyth, A.K. Dietary intakes of professional and semi-professional team
sport athletes do not meet sport nutrition recommendations—A systematic literature review. Nutrients 2019, 11, 1160. [CrossRef]
[PubMed]
2. Biolo, G.; Gastaldelli, A.; Zhang, X.J.; Wolfe, R.R. Protein synthesis and breakdown in skin and muscle: A leg model of amino acid
kinetics. Am. J. Physiol. 1994, 267, E467–E474. [CrossRef] [PubMed]
3. Rennie, M.J.; Wackerhage, H.; Spangenburg, E.E.; Booth, F.W. Control of the size of the human muscle mass. Annu. Rev. Physiol.
2004, 66, 799–828. [CrossRef] [PubMed]
4. Atherton, P.J.; Etheridge, T.; Watt, P.W.; Wilkinson, D.; Selby, A.; Rankin, D.; Smith, K.; Rennie, M.J. Muscle full effect after oral
protein: Time-dependent concordance and discordance between human muscle protein synthesis and mTORC1 signaling. Am. J.
Clin. Nutr. 2010, 92, 1080–1088. [CrossRef]
5. Moran, L.A.; Horton, H.R.; Scrimgeour, K.G.; Perry, M.D. Amino acids and the primary structure of proteins. In Biochemistry São
Paulo: Pearson Education of Brazil; Moran, L.A., Horton, H.R., Scrimgeour, K.G., Perry, M.D., Eds.; University of Toronto: Toronto,
ON, Canada, 2013; pp. 56–85.
6. Nelson, D.L.; Cox, M.M. Amino acids, peptides and proteins. In Principles of Biochemistry, 6th ed.; Nelson, D.L., Cox, M.M., Eds.;
Artmed Publisher: Porto Alegre, Brazil, 2014; pp. 75–114.
Nutrients 2023, 15, 4003 14 of 18

7. Smith, K.; Barua, J.M.; Watt, P.W.; Scrimgeour, C.M.; Rennie, M.J. Flooding with L-[1-13C] leucine stimulates human muscle
protein incorporation of continuously infused L-[1-13C] valine. Am. J. Physiol. 1992, 262, E372–E376. [CrossRef]
8. Wolfe, R.R. Branched-chain amino acids and muscle protein synthesis in humans: Myth or reality? J. Int. Soc. Sports Nutr. 2017,
14, 30. [CrossRef]
9. Reidy, P.T.; Rasmussen, B.B. Role of ingested amino acids and protein in the promotion of resistance exercise-induced muscle
protein anabolism. J. Nutr. 2016, 146, 155–183. [CrossRef]
10. Tang, J.E.; Phillips, S.M. Maximizing muscle protein anabolism: The role of protein quality. Curr. Opin. Clin. Nutr. Metab. Care
2009, 12, 66–71. [CrossRef]
11. Hulmi, J.J.; Lockwood, C.M.; Stout, J.R. Effect of protein/essential amino acids and resistance training on skeletal muscle
hypertrophy: A case for whey protein. Nutr. Metab. 2010, 7, 51. [CrossRef]
12. Stark, M.; Lukaszuk, J.; Prawitz, A.; Salacinski, A. Protein timing and its effects on muscular hypertrophy and strength in
individuals engaged in weight-training. J. Int. Soc. Sports Nutr. 2012, 9, 54. [CrossRef]
13. Cermak, N.M.; Res, P.T.; de Groot, L.C.; Saris, W.H.; van Loon, L.J. Protein supplementation augments the adaptive response of
skeletal muscle to resistance-type exercise training: A meta-analysis. Am. J. Clin. Nutr. 2012, 96, 1454–1464. [CrossRef] [PubMed]
14. Bosse, J.D.; Dixon, B.M. Dietary protein to maximize resistance training: A review and examination of protein spread and change
theories. J. Int. Soc. Sports Nutr. 2012, 9, 42. [CrossRef] [PubMed]
15. Schoenfeld, B.J.; Aragon, A.A.; Krieger, J.W. The effect of protein timing on muscle strength and hypertrophy: A meta-analysis.
J. Int. Soc. Sports Nutr. 2013, 10, 53. [CrossRef] [PubMed]
16. Dideriksen, K.; Reitelseder, S.; Holm, L. Influence of amino acids, dietary protein, and physical activity on muscle mass
development in humans. Nutrients 2013, 5, 852–876. [CrossRef]
17. Hayes, A.; Cribb, P.J. Effect of whey protein isolate on strength, body composition and muscle hypertrophy during resistance
training. Curr. Opin. Clin. Nutr. Metab. Care 2008, 11, 40–44. [CrossRef]
18. Pasiakos, S.M.; McLellan, T.M.; Lieberman, H.R. The effects of protein supplements on muscle mass, strength, and aerobic and
anaerobic power in healthy adults: A systematic review. Sports Med. 2015, 45, 111–131. [CrossRef]
19. Pasiakos, S.M.; Lieberman, H.R.; McLellan, T.M. Effects of protein supplements on muscle damage, soreness and recovery of
muscle function and physical performance: A systematic review. Sports Med. 2014, 44, 655–670. [CrossRef]
20. Miller, P.E.; Alexander, D.D.; Perez, V. Effects of whey protein and resistance exercise on body composition: A meta-analysis of
randomized controlled trials. J. Am. Coll. Nutr. 2014, 33, 163–175. [CrossRef]
21. Skernevičius, J.; Milašius, K.; Raslanas, A.; Dadelienė, R. Sporto treniruotė (Sports training). In Sportininku˛ Gebėjimai ir ju˛ Ugdymas
(Skills and Training of Athletes), 1st ed.; Čepulėnas, A., Saplinskas, J., Paulauskas, R., Eds.; Lithuanian University of Educational
Sciences Press: Vilnius, Lithuania, 2011; pp. 165–217.
22. Duren, D.L.; Sherwood, R.J.; Czerwinski, S.A.; Lee, M.; Choh, A.C.; Siervogel, R.M.; Chumlea, W.C. Body composition methods:
Comparisons and interpretation. J. Diabetes Sci. Technol. 2008, 2, 1139–1146. [CrossRef]
23. Yang, S.W.; Kim, T.H.; Choi, H.M. The reproducibility and validity verification for body composition measuring devices using
bioelectrical impedance analysis in Korean adults. J. Exerc. Rehabil. 2018, 14, 621–627. [CrossRef]
24. Carrion, B.M.; Wells, A.; Mayhew, J.L.; Koch, A.J. Concordance among bioelectrical impedance analysis measures of percent body
fat in athletic young adults. Int. J. Exerc. Sci. 2019, 12, 324–331. [PubMed]
25. Dimitrijevic, M.; Paunovic, V.; Zivkovic, V.; Bolevich, S.; Jakovljevic, V. Body fat evaluation in male athletes from combat sports by
comparing anthropometric, bioimpedance, and dual-energy X-ray absorptiometry measurements. Biomed Res. Int. 2022, 2022,
3456958. [CrossRef] [PubMed]
26. Baranauskas, M.; Jablonskienė, V.; Abaravičius, J.A.; Samsonienė, L.; Stukas, R. Dietary acid-base balance in high-performance
athletes. Int. J. Environ. Res. Public Health 2020, 17, 5332. [CrossRef] [PubMed]
27. Harris, J.; Benedict, F. A Biometric Study of Basal Metabolism in Man; Lippincott: Philadelphia, PA, USA, 1919.
28. Thomas, D.T.; Erdman, K.A.; Burke, L.M. American College of Sports Medicine Joint Position Statement. Nutrition and athletic
performance. Med. Sci. Sports Exerc. 2016, 48, 543–568.
29. Ainsworth, B.E.; Haskell, W.L.; Herrmann, S.D.; Meckes, N.; Basset, D.R.; Todor-Locke, C.; Greer, J.L.; Vezina, J.; Whitt-Glover,
M.C.; Leon, A.S. Compendium of physical activities: A second update of codes and MET values. Med. Sci. Sports Exerc. 2011, 43,
1575–1581. [CrossRef]
30. Deakin, V.; Kerr, D.; Boushey, C. Measuring nutritional status of athletes: Clinical and research perspectives. In Clinical Sports
Nutrition, 5th ed.; Burke, L.M., Deakin, V., Eds.; McGraw-Hill: North Ryde, Australia, 2015; pp. 27–53.
31. Devlin, B.L.; Leveritt, M.D.; Kingsley, M.; Belski, R. Dietary intake, body composition, and nutrition knowledge of Australian
football and soccer players: Implications for sports nutrition professionals in practice. Int. J. Sport Nutr. Exerc. Metab. 2017, 27,
130–138. [CrossRef]
32. Bradley, W.J.; Cavanagh, B.P.; Douglas, W.; Donovan, T.F.; Morton, J.P.; Close, G.L. Quantification of training load, energy intake,
and physiological adaptations during a rugby preseason: A case study from an elite european rugby union squad. J. Strength
Cond. Res. 2015, 29, 534–544. [CrossRef]
33. Bettonviel, A.E.O.; Brinkmans, N.Y.J.; Russcher, K.; Wardenaar, F.C.; Witard, O.C. Nutritional status and daytime pattern of
protein intake on match, post-match, rest and training days in senior professional and youth elite soccer players. Int. J. Sport Nutr.
Exerc. Metab. 2016, 26, 285–293. [CrossRef]
Nutrients 2023, 15, 4003 15 of 18

34. Andrews, M.C.; Itsiopoulos, C. Room for improvement in nutrition knowledge and dietary intake of male football (soccer) players
in Australia. Int. J. Sport Nutr. Exerc. Metab. 2016, 26, 55–64. [CrossRef]
35. Jeoung, B.; Kim, J. Analysis and evaluation of nutritional intake and nutrition quotient of Korean athletes with disabilities in the
Tokyo Paralympic games. Nutrients 2021, 13, 3631. [CrossRef]
36. Elmadfa, I.; Meyer, A.; Nowak, V.; Hasenegger, V.; Putz, P.; Verstraeten, R.; Remaut-DeWinter, A.M.; Kolsteren, P.; Dostálová, J.;
Dlouhý, P.; et al. European Nutrition and Health Report 2009; Forum of Nutrition; Karger Medical and Scientific Publishers: Basel,
Switzerland, 2009; Volume 62, pp. 1–405.
37. Pomerleau, J.; McKee, M.; Robertson, A.; Vaasc, S.; Kadziauskiene, K.; Abaravicius, A.; Bartkeviciute, R.; Pudule, I.; Grinberga, D.
Physical inactivity in the Baltic countries. Prev. Med. 2000, 31, 665–672. [CrossRef]
38. Capling, L.; Beck, K.L.; Gifford, J.A.; Slater, G.; Flood, V.M.; O’Connor, H. Validity of dietary assessment in athletes: A systematic
review. Nutrients 2017, 9, 1313. [CrossRef] [PubMed]
39. Barzda, A.; Bartkevičiūtė, R.; Viseckienė, V.; Abaravičius, A.J.; Stukas, R. Maisto produktu˛ ir patiekalu˛ porciju˛ nuotrauku˛ atlasas (Atlas of
foodstuffs and dishes), Vilnius, Republican Nutrition Center; Vilnius University Faculty of Medicine: Vilnius, Lithuania, 2007; pp. 7–42.
Available online: http://www.smlpc.lt/media/file/Skyriu_info/Metodine_medziaga/Maisto%20prod%20atlasas%202007.pdf
(accessed on 14 June 2020).
40. Sučilienė, S.; Abaravičius, A. Maisto Produktu˛ Sudėtis (Food Product Composition); Ministry of Health of the Republic of Lithuania:
Vilnius, Lithuania, 2002; pp. 10–315.
41. Rodriguez, N.R.; Dimarco, N.M.; Langley, S. Position of the American Dietetic Association, Dietitians of Canada, and the
American College of Sports Medicine: Nutrition and athletic performance. J. Am. Diet. Assoc. 2009, 109, 509–527. [PubMed]
42. Potgieter, S. Sport nutrition: A review of the latest guidelines for exercise and sport nutrition from the American college of sport
nutrition, the International Olympic Committee and the International Society for Sports Nutrition. S. Afr. J. Clin. Nutr. 2013, 26,
6–16. [CrossRef]
43. Kerksick, C.M.; Arent, S.; Schoenfeld, B.J.; Stout, J.R.; Campbell, B.; Wilborn, C.D.; Taylor, L.; Kalman, D.; Smith-Ryan, A.E.;
Kreider, R.B.; et al. International Society of Sports Nutrition position stand: Nutrient timing. J. Int. Soc. Sports Nutr. 2017, 14, 33.
[CrossRef] [PubMed]
44. Aragon, A.A.; Schoenfeld, B.; Wildman, R.; Kleiner, S.; Vandusseldorp, T.; Taylor, L.; Earnest, C.P.; Arciero, P.J.; Wilborn, C.;
Kalman, D.S.; et al. International Society of Sports Nutrition position stand: Diets and body composition. J. Int. Soc. Sports Nutr.
2017, 14, 16. [CrossRef] [PubMed]
45. Mountjoy, M.; Sundgot-Borgen, J.; Burke, L.; Ackerman, K.E.; Blauwet, C.; Constantini, N.; Lebrun, C.; Lundy, B.; Melin, A.;
Meyer, N.; et al. International Olympic Committee (IOC) consensus statement on relative energy deficiency in sport (RED-S):
2018 update. Int. J. Sport Nutr. Exerc. Metab. 2018, 28, 316–331. [CrossRef]
46. International Olympic Committee. IOC consensus statement on sports nutrition 2010. Int. J. Sport Nutr. Exerc. Metab. 2010, 1003,
S3–S4.
47. Kerksick, C.M.; Wilborn, C.D.; Roberts, M.D.; Smith-Ryan, A.; Kleiner, S.M.; Jäger, R.; Collins, R.; Cooke, M.; Davis, J.N.;
Galvan, E.; et al. ISSN exercise & sports nutrition review update: Research & recommendations. J. Int. Soc. Sports Nutr. 2018,
15, 8.
48. Maughan, R.J.; Burke, L.M.; Dvorak, J.; Larson-Meyer, D.E.; Peeling, P.; Phillips, S.M.; Rawson, E.S.; Walsh, N.P.; Garthe, I.;
Geyer, H.; et al. IOC consensus statement: Dietary supplements and the high-performance athlete. Int. J. Sport Nutr. Exerc. Metab.
2018, 28, 104–125. [CrossRef]
49. World Health Organization, Food and Agriculture Organization of the United Nations, United Nations University. Protein and
Amino Acid Requirements in Human Nutrition; Report of a joint FAO/WHO/UNU expert consultation (WHO Technical Report
Series 935). 2007; p. 150. Available online: https://apps.who.int/iris/handle/10665/43411 (accessed on 15 April 2023).
50. Jäger, R.; Kerksick, C.M.; Campbell, B.I.; Cribb, P.J.; Wells, S.D.; Skwiat, T.M.; Purpura, M.; Ziegenfuss, T.N.; Ferrando, A.A.;
Arent, S.M.; et al. International society of sports nutrition position stand: Protein and exercise. J. Int. Soc. Sports Nutr. 2017, 14, 20.
[CrossRef] [PubMed]
51. Mandolfo, S.; Zucchi, A.; Cavalieri D’Oro, L.; Corradi, B.; Imbasciati, E. Protein nitrogen appearance in CAPD patients: What is
the best formula? Nephrol. Dial. Transplant. 1996, 11, 1592–1596. [CrossRef] [PubMed]
52. Folin, O. Laws governing the chemical composition of urine. Am. J. Physiol. 1905, 13, 66–115. [CrossRef]
53. Jeevanandam, M.; Young, D.H.; Ramias, L.; Schiller, W.R. Aminoaciduria of severe trauma. Am. J. Clin. Nutr. 1989, 49, 814–822.
[CrossRef] [PubMed]
54. Maroni, B.J.; Steinman, T.; Mitch, W.E. A method for estimatingnitrogen intake of patients with chronic renal failure. Kidney Int.
1985, 27, 58–65. [CrossRef]
55. Mackenzie, T.A.; Clark, N.G.; Bistrian, B.R.; Flatt, J.P.; Hallowell, E.M.; Blackburn, G.L. A simple method for estimating nitrogen
balance in hospitalized patients: A review and supporting data for a previously proposed technique. J. Am. Coll. Nutr. 1985, 4,
575–581. [CrossRef]
56. Bergstrom, J.; Furst, P.; Alvestrand, A.; Lindholm, B. Protein and energy intake, nitrogen balance and nitrogen losses in patients
treated with continuous ambulatory peritoneal dialysis. Kidney Int. 1993, 44, 1048–1057. [CrossRef]
57. Strengthening the Reporting of Observational Studies in Epidemiology (STROBE). Available online: https://www.strobe-
statement.org/ (accessed on 15 August 2023).
Nutrients 2023, 15, 4003 16 of 18

58. Cohen, J. Statistical Power Aanalysis for the Behavioral Sciences; Routledge Academic: New York, NY, USA, 1988.
59. Sesbreno, E.; Dziedzic, C.E.; Sygo, J.; Blondin, D.P.; Haman, F.; Leclerc, S.; Brazeau, A.-S.; Mountjoy, M. Elite male volleyball
players are at risk of insufficient energy and carbohydrate intake. Nutrients 2021, 13, 1435. [CrossRef]
60. Raymond, C.J.; Dengel, D.R.; Bosch, T.A. Total and segmental body composition examination in collegiate football players
using multifrequency bioelectrical impedance analysis and dual x-ray absorptiometry. J. Strength Cond. Res. 2018, 32, 772–782.
[CrossRef]
61. Rector, R.S.; Rogers, R.; Ruebel, M.; Hinton, P.S. Participation in road cycling vs. running is associated with lower bone mineral
density in men. Metabolism 2008, 57, 226–232. [CrossRef]
62. Domingos, C.; Matias, C.N.; Cyrino, E.S.; Sardinha, L.B.; Silva, A.M. The usefulness of Tanita TBF-310 for body composition
assessment in Judo athletes using a four-compartment molecular model as the reference method. Rev. Assoc. Med. Bras. 2019, 65,
1283–1289. [CrossRef]
63. Reale, R.; Burke, L.M.; Cox, G.R.; Slater, G. Body composition of elite Olympic combat sport athletes. Eur. J. Sport Sci. 2020, 20,
147–156. [CrossRef] [PubMed]
64. Alvero-Cruz, J.R.; Brikis, M.; Chilibeck, P.; Frings-Meuthen, P.; Vico Guzmán, J.F.; Mittag, U.; Michely, S.; Mulder, E.; Tanaka, H.;
Tank, J.; et al. Age-related decline in vertical jumping performance in masters track and field athletes: Concomitant influence of
body composition. Front. Physiol. 2021, 12, 643649. [CrossRef] [PubMed]
65. Domínguez, R.; López-Domínguez, R.; López-Samanes, Á.; Gené, P.; González-Jurado, J.A.; Sánchez-Oliver, A.J. Analysis of sport
supplement consumption and body composition in Spanish elite rowers. Nutrients 2020, 12, 3871. [CrossRef] [PubMed]
66. Ceylan, B.; Gurses, V.V.; Akgul, M.S.; Baydil, B.; Franchini, E. Anthropometric profile, wingate performance and special judo
fitness levels of Turkish Olympic judo athletes. Ido Mov. Culture. J. Martial Arts Anthrop. 2018, 18, 15–20.
67. Rybakova, E.; Shutova, T.; Vysotskaya, T. Sports training of ski jumpers from a springboard based on body composition control
and physical fitness. Int. J. Phys. Educ. Sports Health 2020, 20, 752–758.
68. Zagursky, N.S. Interrelation of indicators of body composition with the effectiveness of competitive activity of highly qualified
biathletes. Theory Pract. Phys. Cult. 2023, 1, 13–16.
69. Lai, Y.-K.; Ho, C.-Y.; Lai, C.-L.; Taun, C.-Y.; Hsieh, K.-C. Assessment of standing multi-frequency bioimpedance analyzer to
measure mody composition of the whole body and limbs in elite male wrestlers. Int. J. Environ. Res. Public Health 2022, 19, 15807.
[CrossRef]
70. Hasan, M.F.; Bahri, S.; Adnyana, I.K. Identification of nutritional status and body composition in weightlifting athlete. Int. J. Phys.
Educ. Sports Health 2021, 21, 2308–2312.
71. Kim, T.H.; Han, J.K.; Lee, J.Y.; Choi, Y.C. The effect of polarized training on the athletic performance of male and female
cross-country skiers during the general preparation period. Healthcare 2021, 9, 851. [CrossRef]
72. Smarkusz-Zarzecka, J.; Ostrowska, L.; Leszczyńska, J.; Orywal, K.; Cwalina, U.; Pogodziński, D. Analysis of the impact of a
multi-strain probiotic on body composition and cardiorespiratory fitness in long-distance runners. Nutrients 2020, 12, 3758.
[CrossRef] [PubMed]
73. Cech, P.; Maly, T.; Mala, L.; Zahalka, F. Body composition of elite youth pentathletes and its gender differences. Sport Sci. 2013, 2,
29–35.
74. Noh, N.M.; Shuhaimi, F.A.; Nor, N.M. Carbohydrate intakes and preferences among endurance athletes in universiti teknologi
MARA Selangor. J. Phys. Conf. Ser. 2020, 1496, 012010.
75. Beermann, B.L.; Lee, D.G.; Almstedt, H.C.; McCormack, W.P. Nutritional intake and energy availability of collegiate distance
runners. J. Am. Coll. Nutr. 2020, 39, 747–755. [CrossRef] [PubMed]
76. Goldenstein, S.; Zaplatosch, M. Energy intake over multiple days of high intensity bike racing: A pilot study. J. Phys. Conf. Ser.
2023, 16, 167.
77. Furber, M.; Pyle, S.; Roberts, M.; Roberts, J. Comparing Acute, high dietary protein and carbohydrate intake on transcriptional
biomarkers, fuel utilisation and exercise performance in trained male runners. Nutrients 2021, 13, 4391. [CrossRef] [PubMed]
78. Burke, L.M. Fuelling strategies to optimize performance: Training high or training low? Scand. J. Med. Sci. Sports 2010, 20, 48–58.
[CrossRef]
79. Morton, J.P.; Croft, L.; Bartlett, J.D.; Maclaren, D.P.M.; Reilly, T.; Evans, L.; McArdle, A.; Drust, B. Reduced carbohydrate
availability does not modulate training-induced heat shock protein adaptations but does upregulate oxidative enzyme activity in
human skeletal muscle. J. Appl. Physiol. 2009, 106, 1513–1521. [CrossRef]
80. Van Proeyen, K.; Szlufcik, K.; Nielens, H.; Ramaekers, M.; Hespel, P. Beneficial metabolic adaptations due to endurance exercise
training in the fasted state. J. Appl. Physiol. 2011, 110, 236–245. [CrossRef]
81. Yeo, W.K.; Paton, C.D.; Garnham, A.P.; Burke, L.M.; Carey, A.L.; Hawley, J.A. Skeletal muscle adaptation and performance
responses to once a day versus twice every second day endurance training regimens. J. Appl. Physiol. 2008, 105, 1462–1470.
[CrossRef]
82. Burke, L.M.; Ross, M.L.; Garvican-Lewis, L.A.; Welvaert, M.; Heikura, I.A.; Forbes, S.G.; Mirtschin, J.G.; Cato, L.E.; Strobel, N.;
Sharma, A.P.; et al. Low carbohydrate, high fat diet impairs exercise economy and negates the performance benefit from
intensified training in elite race walkers. J. Physiol. 2017, 595, 2785–2807. [CrossRef] [PubMed]
Nutrients 2023, 15, 4003 17 of 18

83. Gejl, K.D.; Thams, L.B.; Hansen, M.; Rokkedal-Lausch, T.; Plomgaard, P.; Nybo, L.; Larsen, F.J.; Cardinale, D.A.; Jensen, K.;
Holmberg, H.C.; et al. No superior adaptations to carbohydrate periodization in elite endurance athletes. Med. Sci. Sports Exerc.
2017, 49, 2486–2497. [CrossRef] [PubMed]
84. Bilsborough, J.C.; Greenway, K.; Opar, D.; Livingstone, S.; Cordy, J.; Coutts, A.J. The accuracy and precision of DXA for assessing
body composition in team sport athletes. J. Sports Sci. 2014, 32, 1821–1828. [CrossRef] [PubMed]
85. Tooley, E.; Bitcon, M.; Briggs, M.A.; West, D.J.; Russell, M. Estimates of energy intake and expenditure in professional rugby
league players. Int. J. Sports Sci. Coach. 2015, 10, 551–560. [CrossRef]
86. Jenner, S.L.; Trakman, G.; Coutts, A.; Kempton, T.; Ryan, S.; Forsyth, A.; Belski, R. Dietary intake of professional Australian
football athletes surrounding body composition assessment. J. Int. Soc. Sports Nutr. 2018, 15, 43. [CrossRef]
87. Gibson-Smith, E.; Storey, R.; Ranchordas, M. Dietary intake, body composition and iron status in experienced and elite climbers.
Front. Nutr. 2020, 7, 122. [CrossRef]
88. Nunes, C.L.; Matias, C.N.; Santos, D.A.; Morgado, J.P.; Monteiro, C.P.; Sousa, M.; Minderico, C.S.; Rocha, P.M.; St-Onge, M.-P.;
Sardinha, L.B.; et al. Characterization and comparison of nutritional intake between preparatory and competitive phase of highly
trained athletes. Medicina 2018, 54, 41. [CrossRef]
89. Conejos, C.; Giner, A.; Mañes, J.; Soriano, J.M. Energy and nutritional intakes in training days of soccer players according to their
playing positions. Arch. Med. Deporte 2011, 28, 29–35.
90. Potgieter, S.; Visser, J.; Croukamp, I.; Markides, M.; Nascimento, J.; Scott, K. Body composition and habitual and match-day
dietary intake of the FNB Maties Varsity Cup rugby players. S. Afr. J. Sports Med. 2014, 26, 35–43. [CrossRef]
91. Molina-López, J.; Molina, J.M.; Chirosa, L.J.; Florea, D.; Sáez, L.; Jiménez, J.; Planells, P.; Pérez de la Cruz, A.; Planells, E.
Implementation of a nutrition education program in a handball team; consequences on nutritional status. Nutr. Hosp. 2013, 28,
1065–1076.
92. Valliant, M.W.; Pittman Emplaincourt, H.; Wenzel, R.K.; Garner, B.H. Nutrition education by a registered dietitian improves
dietary intake and nutrition knowledge of a NCAA female volleyball team. Nutrients 2012, 4, 506–516. [CrossRef]
93. Baranauskas, M.; Tubelis, L.; Stukas, R.; Švedas, E.; Samsonienė, L.; Karanauskienė, D. Nutrition status of high performance
rowers, canoeists and kayakers. Balt. J. Sport Health Sci. 2014, 1, 16–26. [CrossRef]
94. Bukhari, S.S.; Phillips, B.E.; Wilkinson, D.J.; Limb, M.C.; Rankin, D.; Mitchell, W.K.; Kobayashi, H.; Greenhaff, P.L.; Smith, K.;
Atherton, P.J. Intake of low-dose leucine-rich essential amino acids stimulates muscle anabolism equivalently to bolus whey
protein in older women at rest and after exercise. Am. J. Physiol. Endocrinol. Metab. 2015, 308, E1056–E1065. [CrossRef] [PubMed]
95. Greenhaff, P.L.; Karagounis, L.G.; Peirce, N.; Simpson, E.J.; Hazell, M.; Layfield, R.; Wackerhage, H.; Smith, K.; Atherton, P.; Selby,
A.; et al. Disassociation between the effects of amino acids and insulin on signaling, ubiquitin ligases, and protein turnover in
human muscle. Am. J. Physiol. Endocrinol. Metab. 2008, 295, E595–E604. [CrossRef]
96. Moore, D.R. One size doesn’t fit all: Postexercise protein requirements for the endurance athlete. Am. J. Clin. Nutr. 2020, 112,
249–250. [CrossRef] [PubMed]
97. Blomstrand, E.; Eliasson, J.; Karlsson, H.K.R.; Kohnke, R. Branched-chain amino acids activate key enzymes in protein synthesis
after physical exercise. J. Nutr. 2006, 136, 269S–273S. [CrossRef] [PubMed]
98. Santos, C.S.; Nascimento, F.E.L. Isolated branched-chain amino acid intake and muscle protein synthesis in humans: A biochemical
review. Einstein 2019, 17, eRB4898. [CrossRef]
99. Hyde, R.; Taylor, P.M.; Hundal, H.S. Amino acid transporters: Roles in amino acid sensing and signalling in animal cells. Biochem.
J. 2003, 373, 1–18. [CrossRef]
100. Szmelcman, S.; Guggenheim, K. Interference between leucine, isoleucine and valine during intestinal absorption. Biochem. J. 1996,
100, 7–11. [CrossRef]
101. Goron, A.; Moinard, C. Amino acids and sport: A true love story? Amino Acids 2018, 50, 969–980. [CrossRef]
102. De Souza, M.J.; Williams, N.I.; Nattiv, A.; Joy, E.; Misra, M.; Loucks, A.B.; Matheson, G.; Olmsted, M.P.; Barrack, M.; Mallinson,
R.J.; et al. Misunderstanding the female athlete triad: Refuting the IOC consensus statement on relative energy deficiency in sport
(RED-S). Br. J. Sports Med. 2014, 48, 1461–1465. [CrossRef] [PubMed]
103. Holtzman, B.; Ackerman, K.E. Recommendations and nutritional considerations for female athletes: Health and performance.
Sports Med. 2021, 51, 43–57. [CrossRef]
104. Nattiv, A.; Loucks, A.B.; Manore, M.M.; Sanborn, C.F.; Sundgot-Borgen, J.; Warren, M.P. American College of Sports Medicine
position stand. The female athlete triad. Med. Sci. Sports Exerc. 2007, 39, 1867–1882. [PubMed]
105. Ackerman, K.E.; Holtzman, B.; Cooper, K.M.; Flynn, E.F.; Bruinvels, G.; Tenforde, A.S.; Popp, K.L.; Simpkin, A.J.; Parziale, A.L.
Low energy availability surrogates correlate with health and performance consequences of relative energy deficiency in sport. Br.
J. Sports Med. 2019, 53, 628–633. [CrossRef] [PubMed]
106. De Souza, M.J.; Nattiv, A.; Joy, E.; Misra, M.; Williams, N.I.; Mallinson, R.J.; Gibbs, J.C.; Olmsted, M.; Goolsby, M.;
Matheson, G.; et al. 2014 female athlete triad coalition consensus statement on treatment and return to play of the female
athlete triad: 1st international conference held in San Francisco, California, May 2012 and 2nd international conference held in
Indianapolis, Indiana, May 2013. Br. J. Sports Med. 2014, 48, 289. [PubMed]
107. Al-Khelaifi, F.; Diboun, I.; Donati, F.; Botrè, F.; Alsayrafi, M.; Georgakopoulos, C.; Suhre, K.; Yousri, N.A.; Elrayess, M.A. A pilot
study comparing the metabolic profiles of elite-level athletes from different sporting disciplines. Sports Med. Open. 2018, 4, 2.
[CrossRef]
Nutrients 2023, 15, 4003 18 of 18

108. Livingstone, M.B.; Prentice, A.M.; Coward, W.A.; Strain, J.J.; Black, A.E.; Davies, P.S.; Stewart, C.M.; McKenna, P.G.;
Whitehead, R.G. Validation of estimates of energy intake by weighed dietary records and diet history in children and adolescents.
Am. J. Clin. Nutr. 1992, 56, 29–35. [CrossRef] [PubMed]
109. Prentice, A.M.; Black, A.E.; Coward, W.A.; Davies, H.L.; Goldberg, G.R.; Murgatroyd, P.R.; Ashford, J.; Sawyer, M.;
Whitehead, R.G. High levels of energy expenditure in obese women. Br. Med. J. 1986, 292, 983–987. [CrossRef]
110. Hill, R.J.; Davies, P. The validity of self-reported energy intake as determined using the doubly labelled water technique. Br. J.
Nutr. 2001, 85, 415–430. [CrossRef]
111. Westerterp, K.R.; Saris, W.H.M.; van Es, M.; ten Hoor, F. Use of the doubly labeled water technique in humans during heavy
sustained exercise. J. App. Physiol. 1986, 61, 2162–2167. [CrossRef]
112. Haggarty, P.; McGaw, B.A.; Maughan, R.J.; Fenn, C. Energy expenditure of elite female athletes measured by the doubly labeled
water method. Proc. Nutr. Soc. 1988, 47, 35.
113. Magkos, F.; Yannakoulia, M. Methodology of dietary assessment in athletes: Concepts and pitfalls. Curr. Opin. Clin. Nutr. Metab.
Care 2003, 6, 539–549. [CrossRef] [PubMed]
114. Braakhuis, A.J.; Meredith, K.; Cox, G.R.; Hopkins, W.G.; Burke, L.M. Variability in estimation of self-reported dietary intake
data from elite athletes resulting from coding by different sports dietitians. Int. J. Sport Nutr. Exerc. Metab. 2003, 13, 152–165.
[CrossRef]
115. Ward, K.D.; Hunt, K.M.; Burstyne Berg, M.; Slawson, D.A.; Vukadinovich, C.M.; McClanahan, B.S.; Clemens, L.H. Reliability and
validity of a brief questionnaire to assess calcium intake in female collegiate athletes. Int. J. Sport Nutr. Exerc. Metab. 2004, 14,
209–221. [CrossRef]
116. Paton, N.I.J.; Macallan, D.C.; Jebb, S.A.; Pazianas, M.; Griffin, G.E. Dual-energy X-ray absorptiometry results differ between
machines. Lancet 1995, 346, 899–900. [CrossRef]
117. Nana, A.; Slater, G.J.; Stewart, A.D.; Burke, L.M. Methodology review: Using dual-energy X-ray absorptiometry (DXA) for the
assessment of body composition in athletes and active people. Int. J. Sport Nutr. Exerc. Metab. 2015, 25, 198–215. [CrossRef]
118. Nana, A.; Slater, G.J.; Hopkins, W.G.; Halson, S.L.; Martin, D.T.; West, N.P.; Burke, L.M. Importance of standardized DXA protocol
for assessing physique changes in athletes. Int. J. Sport Nutr. Exerc. Metab. 2016, 26, 259–267. [CrossRef]
119. Deutz, R.C.; Benardot, D.; Martin, D.E.; Cody, M.M. Relationship between energy deficits and body composition in elite female
gymnasts and runners. Med. Sci. Sports Exerc. 2000, 32, 659–668. [CrossRef]
120. Bilsborough, J.C.; Kempton, T.; Greenway, K.; Cordy, J.; Coutts, A.J. Longitudinal changes and seasonal variation in body
composition in professional Australian football players. Int. J. Sports Physiol. Perform. 2017, 12, 10–17. [CrossRef]
121. Bartlett, J.D.; Hatfield, M.; Parker, B.B.; Roberts, L.A.; Minahan, C.; Morton, J.P.; Thornton, H.R. DXA-derived estimates of energy
balance and its relationship with changes in body composition across a season in team sport athletes. Eur. J. Sport Sci. 2020, 20,
859–867. [CrossRef]
122. Milsom, J.; Naughton, R.; O’Boyle, A.; Iqbal, Z.; Morgans, R.; Drust, B.; Morton, J.P. Body composition assessment of English
Premier League soccer players: A comparative DXA analysis of first Team, U21 and U18 Squads. J. Sports Sci. 2015, 33, 1799–1806.
[CrossRef] [PubMed]
123. Alburquerque-Sendín, F.; Martín-Vallejo, F.J.; García-Talavera, P.; Martín Gómez, M.E.; Santos del Rey, M. Comparison of fat mass
and fat-free mass between Anthropometry, BIA and DEXA in young females: Are methods really interchangeable? Eur. J. Anat.
2020, 14, 133141.
124. Syed-Abdul, M.M.; Dhwani, S.; Jason, D.W. Effects of self-implemented carbohydrate cycling and moderate to high intensity
resistance exercise on body fat in body builders. Gazz. Med. Ital. Archivio Sci. Med. 2019, 178, 221–224. [CrossRef]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.