Ethel Machergiany*

ethel17@gmail.com

Jorge Soares**

jfpsoares@utad.pt

Luíz Azevedo*

jfpsoares@utad.pt

Catarina Abrantes**

aabrantes@utad.pt

Ana Barros***

abarros@utad.pt

Maria Paula Mota**

mpmota@utad.pt

*Research Center for Sports Sciences, Health and Human Development (CIDESD)

University of Trás-os-Montes and Alto Douro, Vila Real

**Research Center for Sports Sciences, Health and Human Development (CIDESD)

and Department of Sport of Science Exercise and Health, School

of Life and Environmental Sciences, (ECVA)

of University of Trás-os-Montes and Alto Douro, Vila Real

***Centre for the Research and Technology

of Agro-Environmental and Biological Sciences (CITAB)

of University of Trás-os-Montes and Alto Douro, Vila Real

(Portugal)

Reception: 02/14/2025 - Acceptance: 03/20/2025

1st Review: 02/25/2025 - 2nd Review: 03/17/2025

Accessible document. Law N° 26.653. WCAG 2.0

Suggested reference: Machergiany, E., Soares, J., Azevedo, L., Abrantes, C., Barros, A., & Mota, M.P. (2025). Plant Protein and Strength Training: Impacts on Plasma Leucine Concentrations. Lecturas: Educación Física y Deportes, 30(323), 105-119. https://doi.org/10.46642/efd.v30i323.8136

 

Abstract

    Physical exercise, especially strength training, has health benefits, including increased muscle mass. Adequate protein intake was essential to optimise these effects, especially leucine, an essential amino acid. Although proteins of animal origin have been considered superior, plant proteins, when combined, could also be effective. This study aimed to evaluate the effects of a protein supplement derived from immature cowpea pods on plasma leucine concentration in young adults after a strength training session. Ten healthy men took part in the study, performing strength training under four conditions: control, exercise, supplementation and supplementation with exercise. Blood samples were taken to analyse leucine. The supplementation consisted of 150g of mashed cowpea pods, with an estimated protein content of between 15g and 18g. Leucine levels varied significantly between conditions. Exercise alone maintained levels for longer, while supplementation resulted in an earlier but smaller increase. The combination of supplementation and exercise showed a trend of progressive reduction, suggesting that the vegetable protein may not have provided enough leucine to optimise the training response. It was concluded that cowpea protein supplementation did not significantly increase plasma leucine after training, possibly due to slower digestion and a less complete amino acid profile. Additional strategies, such as combining with other amino acids or carbohydrates, may have been necessary to optimise the use of plant proteins in strength training.

    Keywords: Protein supplementation. Vegetable protein. Strength training. Plasma leucine.

 

Resumo

    O exercício físico, especialmente o treino de força, é benéfico à saúde, incluindo o aumento da massa muscular. A ingestão adequada de proteínas foi essencial para otimizar esses efeitos, especialmente a leucina, um aminoácido essencial. Embora as proteínas de origem animal tenham sido consideradas superiores, as proteínas vegetais, quando combinadas, também puderam ser eficazes. Este estudo teve como objetivo avaliar os efeitos de um suplemento proteico derivado de vagens imaturas de feijão-caupi sobre a concentração plasmática de leucina em jovens adultos após uma sessão de treino de força. Dez homens saudáveis participaram do estudo, realizando treino de força sob quatro condições: controle, exercício, suplementação e suplementação com exercício. Amostras de sangue foram coletadas para análise da leucina. A suplementação consistiu em 150g de vagens de feijão-caupi amassadas, com conteúdo proteico estimado entre 15g e 18g. Os níveis de leucina variaram significativamente entre as condições. O exercício, por si só, manteve os níveis por mais tempo, enquanto a suplementação resultou em um aumento precoce, porém menor. A combinação de suplementação e exercício demonstrou uma tendência de redução progressiva, sugerindo que a proteína vegetal pode não ter fornecido leucina suficiente para otimizar a resposta ao treino. Concluiu-se que a suplementação com proteína de feijão-caupi não aumentou significativamente a leucina plasmática após o treino, possivelmente devido à digestão mais lenta e a um perfil de aminoácidos menos completo. Estratégias adicionais, como a combinação com outros aminoácidos ou carboidratos, podem ter sido necessárias para otimizar o uso de proteínas vegetais no treino de força.

    Unitermos: Suplementação de proteínas. Proteína vegetal. Treinamento de força. Leucina plasmática.

 

Resumen

    El ejercicio físico, especialmente el entrenamiento de fuerza, es beneficioso para la salud, incluido el aumento de masa muscular. Una ingesta adecuada de proteínas es esencial para optimizar estos efectos, especialmente la leucina, un aminoácido esencial. Aunque se ha comprobado que las proteínas de origen animal son superiores, las proteínas vegetales, combinadas, también podrían ser eficaces. Este estudio evaluó los efectos de un suplemento proteico derivado de vainas inmaduras de caupí sobre la concentración plasmática de leucina en adultos jóvenes tras una sesión de entrenamiento de fuerza. Participaron diez hombres sanos realizando entrenamiento de fuerza en cuatro condiciones: control, ejercicio, suplementación y suplementación con ejercicio. Se tomaron muestras de sangre para analizar la leucina. La suplementación consistió en 150g de puré de vainas de caupí, con un contenido estimado de proteínas de entre 15g y 18g. Los niveles de leucina variaron significativamente entre condiciones. El ejercicio por sí solo mantuvo los niveles durante más tiempo, mientras que la suplementación produjo aumento más temprano pero menor. La combinación de suplementación y ejercicio mostró una tendencia a la reducción progresiva, lo que sugiere que la proteína vegetal puede no haber proporcionado suficiente leucina para optimizar la respuesta al entrenamiento. Se concluyó que la suplementación con proteína de caupí no aumentaba significativamente la leucina plasmática tras el entrenamiento, posiblemente debido a una digestión más lenta y a un perfil de aminoácidos menos completo. Pueden haber sido necesarias estrategias adicionales, como la combinación con otros aminoácidos o hidratos de carbono.

    Palabras clave: Suplementos proteicos. Proteína vegetal. Entrenamiento de fuerza. Leucina plasmática.

 

Lecturas: Educación Física y Deportes, Vol. 30, Núm. 323, Abr. (2025)

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Introduction 

 

    Regular exercise, especially strength training, has been widely recognised for its health benefits, including increasing muscle mass and reducing the risk of chronic non-communicable diseases (Anderson, & Durstine, 2019; Caspersen et al., 1985; Ravelli et al., 2022). To optimise these benefits, adequate protein intake is essential, since muscle hypertrophy depends on the balance between muscle protein synthesis and degradation (Stokes et al., 2018; Tipton, & Ferrando, 2008). In this context, the International Society of Sports Nutrition (ISSN) recommends a protein intake of 1.4 to 2.0 g/kg of body weight for active individuals seeking to gain muscle mass, emphasising the importance not only of quantity, but also of the quality and distribution of proteins throughout the day (Jäger et al., 2017; Kerksick et al., 2018).

 

    Among the factors that influence protein quality, leucine emerges as a key essential amino acid, acting as a key signalling agent for the activation of the mTOR pathway, which promotes muscle growth (Barrantes-Silman et al., 2023; Yasuda et al., 2020). Although animal proteins, such as whey proteins, are traditionally considered superior due to their high leucine content and biological value, plant proteins can also be effective when combined strategically to provide a complete profile of essential amino acids (Antonio et al., 2020; Kerksick et al., 2021). This approach is particularly relevant since recent studies have shown that, despite their lower digestibility, vegetable proteins can promote adaptations to strength training, provided they are consumed in adequate quantities. (Kerksick et al., 2021; Volek et al., 2013)

 

    This connection between plant proteins and strength training becomes even more relevant when it is considered that resistance exercise is a powerful stimulus for muscle hypertrophy, but its effects are maximised when associated with an adequate protein intake, especially in terms of the amount and timing of consumption (Krzysztofik et al., 2019; Morocho-Quinchuela et al., 2023). Therefore, combining resistance exercise with the intake of leucine-rich plant proteins, such as those found in legumes and whole grains, can be a viable strategy for individuals seeking muscle gains, especially for those who prefer or require plant-based diets (Langyan et al., 2021). Therefore, understanding the role of plant proteins and leucine in the context of strength training is fundamental to developing effective nutritional strategies that meet the needs of different population groups. (Trapp et al., 2010; Willett et al., 2019)

 

    Given these dynamics, the SoilRec4+Health project developed a supplement (Puree) from immature cowpea pods, with the aim of evaluating its effects on leukaemia in young adults after strength training. This study aimed to evaluate the effects of a protein supplement derived from immature cowpea pods on plasma leukaemia in young adults after a strength training session.

 

Methods 

 

Ethical consideration 

 

    This study was approved by the Human Research Ethics Committee (Reference number: NORTE-01-0145-FEDER-000083) and conducted between March and May 2023 at the University of Trás-os-Montes and Alto Douro.

 

Sample 

 

    Ten healthy men (mean ± SD: 26 ± 7.4 years; 65 ± 8.2 kg; 177.5 ± 6.5 centimeters), free of endocrine cardiovascular diseases or other metabolic diseases through self-reported anamnesis were eligible to participate in this study. Participants performed 4 ± 1.1 days a week of different types of physical exercise (soccer, strength training, running, volleyball, etc.) with an average weekly duration of 324 ± 202 minutes, but were not involved in any structured progressive exercise training regimen or following restrictive diets.

The inclusion criteria for the study were defined to ensure homogeneity in the sample. The study included healthy men over the age of 18 who regularly exercised (at least three times a week). The participants had to be available to attend all the experimental sessions and not have suffered any musculoskeletal injuries in the last six months. The exclusion criteria aimed to eliminate factors that could interfere with the results of the study. Individuals diagnosed with metabolic, cardiovascular or musculoskeletal diseases, smokers, people who abuse alcohol, and those taking medication that affects protein metabolism, such as steroids or corticosteroids, were excluded. In addition, participants who were involved in diet or supplementation programs unrelated to the study or who had medical conditions that prevented them from practicing strength exercises were excluded.

 

    The characteristics of the participants are shown in Table 1. Participants were informed about the possible risks of the experimental procedures before providing informed consent and signing the form. Throughout the research, the participants were monitored by three Sports Professionals.

 

Age (years)	Weight (Kg)	Height (Centimeters)	Exercise Time per Week (Minutes)	Weekly Frequency of Physical Exercise (Days)
26 ± 7,4	65 ± 8,2	177,5 ± 6,5	324 ± 202	4 ± 1,1

Note: Values are presented as means ± SD. Source: Research data

 

Protocol for Determining One-Repetition Maximum (1RM) 

 

    All 1RM assessment sessions were scheduled at a consistent time of day for each participant, based on their individual availability. The 1RM evaluation protocol commenced with a general warm-up, which included a 5-minute treadmill walk at a speed of 4.5 km/h, followed by dynamic mobility exercises utilizing body weight or elastic resistance bands. The mobility exercises comprised the following:

 

Series	Repetitions	Exercise
1	10	Alternating side-to-side squats
1	10	Bodyweight squats
1	10	Narrow-stance squats
1	10	Low row using a resistance band;
1	10	Internal shoulder rotation with a resistance band
1	10	External shoulder rotation with a resistance band.

Source: Research data

 

    The 1RM assessment involved progressively increasing loads until the participant reached their maximal lift capacity. Participants were permitted up to 6 attempts per exercise (Chagas et al., 2005; Mayhew et al., 2008), with rest intervals of 3 to 5 minutes between attempts (Mayhew et al., 2008). The test began with a submaximal load, which was incrementally increased with each subsequent attempt. The 1RM value was recorded when the participant failed to complete the movement with proper form, with the weight from the last successful attempt being documented. The test was terminated if any deviation from the standard movement execution occurred. No external assistance was allowed during the test. (Chagas et al., 2005)

 

Strength Training (ST) Protocol 

 

    Prior to each ST session, participants performed a light warm-up consisting of a 5-minute walk on a treadmill at 4.5 km/h. The ST protocol, conducted at two different times in the study, involved the exercises bench press, deadlift, lat pull-down, and leg press. Each session consisted of 3 sets of 8-10 repetitions at 75% of 1RM, with 45 seconds of rest between sets and 1 minute between exercises. ST sessions were scheduled at the same time of day for each participant, typically in the morning following an overnight fast (~8:00). If a session was missed, it was immediately rescheduled for the following day.

 

Study Protocol 

 

    One week after the 1RM measurements, participants were subjected to four different conditions across different weeks. For each condition, participants were instructed to avoid strenuous exercise in the 48 hours preceding and to maintain an overnight fast while following their normal dietary patterns throughout the study. The four situations tested were:

 

Situation	Description
Control (C)	Participants arrived at the laboratory and remained at rest throughout the protocol.
Exercise (E)	Participants performed the ST session based on the previously collected 1RM measurements.
Supplementation (S)	Participants ingested 150g of supplementation and remained at rest.
Supplementation + Exercise (SE)	Participants ingested 150g of supplement and immediately after ingestion, performed the ST protocol.

Note: C = Control E = Exercise S = Supplementation SE = Supplementation + Exercise. Source: Research data

 

    All laboratory visits and conditions were separated by 7 days. For each condition, capillary blood samples (~120 μL) were collected at different times: baseline (pre = 0), 45 minutes post-intervention (post 1), and 90 minutes post-intervention (post 2), using an Accu-Chek Safe-T-Pro-Plus lancing device. The samples were collected into capillary tubes containing heparin and stored in Eppendorf microtubes (1.5 mL) at -20°C for subsequent total amino acid analysis. The study design is illustrated in the following figure.

 

Source: Authors

 

Nutritional facts of Cowpea Puree 

 

    The immature pods of the cowpea are a significant source of vegetable protein, which makes them a valuable addition to the human diet. According to Machado et al. (2017), the protein content in fresh pods can vary, but is generally in the range of 10 to 12 g of protein per 100 g of pod.  This variation is influenced by factors such as the plant variety and growing conditions. In the context of the study, the dose ingested by the participants was 150 g of immature pods. Based on the information available, the protein content for this portion can be estimated at between 15 g and 18 g. This calculation is carried out as follows: for a protein content of 10 g per 100 g, the amount of protein in 150 g would be 10g/100g×150g≈15g per portion. (Machado et al., 2017)

 

Amino Acid Determination 

 

    Amino acid determination was performed using High-Performance Liquid Chromatography with Fluorescence Detection (HPLC-FLD).

 

Statistical analysis 

 

    For statistical analysis, the SPSS (Statistical Package for the Social Sciences) software was used with a significance level of p < 0.05. Descriptive statistics were performed, including mean, standard deviation, and percentage. Normality was assessed using the Kolmogorov-Smirnov test. Data skewness and kurtosis were evaluated to identify and exclude outliers. Considering the normal distribution of variables, repeated measures MANOVA was used to compare different groups and time points.

 

Results 

 

    The values for the percentages of leucine in the blood are shown in Figure 2. Leucine levels showed significant differences between the groups over time (p=0.036 and p=0.259), indicating distinct patterns of variation between the experimental conditions. In the Control Condition (C), a peak of 4.2 per cent was observed at post 1, which remained stable at post 2. In the Exercise Condition (EC), the leucine peak was reached at post 2 (4.1 per cent), while in the Supplementation Condition (SC), the peak was observed at post 1 (2.9 per cent). On the other hand, in the Supplementation + Exercise Condition (SE), leucine levels tended to decrease over the time points, with values of 2.4%, 2.1% and 2.0%, respectively.

 

Source: Authors

 

Discussion 

 

    The results of this study showed that supplementation with vegetable protein derived from cowpea pods, associated with strength training, did not promote a significant increase in plasma leucine concentrations. Leucine, a branched-chain amino acid (BCAA), is recognised as a key regulator of muscle protein synthesis (MPS), activating signalling pathways such as mTOR, which are essential for muscle hypertrophy and recovery (Anthony et al., 2001; Garlick, 2005). However, the lower leucine concentration observed in the group that combined supplementation and exercise (SE) suggests that the plant protein used may not be as effective as animal proteins, such as whey, in raising postprandial leukaemia (Boirie, Gachon, et al., 1997; Pennings et al., 2011). This finding is consistent with previous studies highlighting the importance of protein digestibility and absorption speed in maximising leucine availability in plasma. (Gorissen, Trommelen, Kouw, Holwerda et al., 2020; Tang et al., 2009)

 

    Leucine is particularly important because it acts as a metabolic signalling agent, stimulating MPS after exercise. Studies show that the ingestion of rapidly digestible proteins, such as whey, results in a rapid and pronounced peak of leucine in the plasma, which is crucial for activating the mTOR pathway and promoting protein synthesis (Anthony et al., 2001; Churchward-Venne et al., 2014). In contrast, the vegetable protein used in this study, derived from cowpeas, may be more slowly digested, resulting in a gradual release of amino acids and, consequently, a lower concentration of leucine in the plasma (Boirie, Dangin, et al., 1997; Gorissen, Trommelen, Kouw, Holwerda et al., 2020). This difference in absorption kinetics may explain why vegetable protein supplementation was unable to significantly raise leucine levels, especially in the SE group.

 

    In addition, the lower leucine concentration observed in the SE group may be related to the increased oxidation of BCAAs during exercise. Leucine, along with isoleucine and valine, is oxidised to provide energy during physical exertion, especially under fasting or high-intensity conditions (Harper et al., 1984; Shimomura et al., 2004). This process may have reduced the availability of leucine in plasma, limiting its ability to stimulate MPS after exercise. Previous studies suggest that supplementation with BCAAs can help compensate for this oxidation, but the effectiveness depends on the quality and quantity of amino acids supplied (Churchward-Venne et al., 2014; Shimomura et al., 2004). Therefore, the vegetable protein used may not have provided enough leucine to compensate for its oxidation during exercise.

 

    The findings of this study are in line with the definition of physical activity and exercise proposed by Caspersen et al. (1985), who emphasise the distinction between general physical activity and structured exercise. According to the authors, the effectiveness of exercise on protein synthesis depends not only on intensity and frequency, but also on the nutritional composition that accompanies it. This differentiation is relevant to interpreting the results of the present study, as the lower plasma leucine concentration observed may be related to the lower efficiency of plant proteins in providing the necessary substrates for activating the mTOR pathway. (Caspersen et al., 1985)

 

    Another factor that may have contributed to the results observed is the amino acid profile of the plant protein. Plant proteins, such as those derived from cowpeas, often have a less complete amino acid profile compared to animal proteins, which may limit their ability to stimulate MPS (Gorissen et al., 2018; van Vliet et al., 2015).

 

    The findings of this study are corroborated by Truong (1996), who emphasize the variation in the amino acid profile of vegetable proteins, which influences the bioavailability of leucine. This limitation may explain the lower plasma concentration observed in the SE group, since vegetable proteins, such as cassava, are less efficient at stimulating protein synthesis (Truong, 1996). This reinforces the need for complementary strategies, such as the addition of isolated leucine, to optimize the anabolic response to exercise.

 

    Leucine is the most critical amino acid for the activation of the mTOR pathway, and its relative concentration in the protein is a determining factor for the anabolic response (Anthony et al., 2001; Garlick, 2005). Therefore, the lower concentration of leucine in plant protein may explain why supplementation did not result in a significant increase in leukaemia, especially when combined with exercise.

 

    Despite the limitations observed, the results suggest that plant protein supplementation can play a complementary role in nutritional strategies aimed at muscle recovery. Recent studies indicate that combining plant proteins with carbohydrates or supplementing with isolated leucine can improve the anabolic response, especially in populations that follow plant-based diets (Gorissen, Trommelen, Kouw, Kouw et al., 2020; Kerksick et al., 2021). In addition, the inclusion of plant proteins in the diet can bring additional benefits, such as reducing environmental impact and promoting cardiovascular health, which justifies exploring strategies to optimise their use in the sports context (Tilman, & Clark, 2014; Willett et al., 2019).

 

    New evidence suggests that supplementing with isolated leucine or combining plant proteins with other essential amino acids may be an effective strategy for improving the anabolic response in individuals who consume plant proteins. A recent study by Zhao et al. (2024) showed that adding leucine to plant proteins significantly increased muscle protein synthesis in athletes, comparable to the effects observed with animal proteins. Another study by Pinckaers et al. (2021) reinforced this idea, showing that combining plant proteins with carbohydrates or essential amino acids can improve the availability of leucine in plasma, optimising the post-exercise anabolic response. In addition, Berrazaga et al. (2019) highlighted that supplementation with BCAAs can compensate for leucine deficiencies in plant proteins, improving muscle recovery and reducing fatigue in athletes, especially when combined with high-intensity exercise. These approaches may be particularly useful for compensating for the limitations of plant proteins, such as the one used in this study.

 

Conclusion 

 

    The results of this study indicated that leucine levels in the blood varied differently between the experimental conditions over time. Exercise alone seemed to sustain leukaemia for a longer period, while supplementation with vegetable protein resulted in an earlier but less significant increase. However, when supplementation was combined with exercise, leucine levels showed a progressive downward trend, suggesting that protein derived from cowpeas may not provide sufficient availability of this amino acid to optimise physiological responses to strength training.

 

    This study has some limitations that should be considered. The small sample size may restrict the generalisability of the findings. In addition, the absence of a detailed assessment of the participants' dietary intake prevents a broader understanding of the dietary factors that may have influenced the results. Finally, the use of a single vegetable protein source makes direct comparisons with other protein sources difficult. Future studies should address these limitations and explore strategies for potentiating the effects of plant-based supplementation in the context of strength training.

 

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Lecturas: Educación Física y Deportes, Vol. 30, Núm. 323, Abr. (2025)