Sports nutrition supplements, high-protein energy bars, and meal replacement protein-based solid beverages are widely consumed by athletes and fitness enthusiasts to restore performance after exercise and to optimize training outcomes (1). To improve the palatability of these processed foods and beverages, non-dairy creamer is frequently incorporated to enhance taste, texture, and overall sensory appeal (2, 3). Specifically, the addition of non-dairy creamer provides a thick and milk fat-like mouthfeel (3). In addition, its convenient storage, stable quality, and low production cost have driven its rapid global expansion across the food industry. The major component in non-dairy creamer responsible for generating this creamy mouthfeel is hydrogenated vegetable oil (4).

However, hydrogenation of vegetable oils introduces potential health concerns. Partial hydrogenation produces trans fats, which are well known to exert adverse metabolic and cardiovascular consequences (5). Full hydrogenation eliminates trans fats, producing fully hydrogenated vegetable oil-based non-dairy creamer (FHVO-NDC), which is primarily composed of saturated fats (6). Growing evidence has suggested that excessive intake of saturated fats may increase the risk of metabolic syndrome (7, 8) and promote chronic low-grade inflammation (9). Importantly, metabolic syndrome has been linked to reductions in muscle mass and strength across multiple studies (10, 11), while chronic inflammation has been associated with impaired athletic performance and delayed recovery (12). Furthermore, consumption of non-dairy creamer has been associated with obesity risk, gut microbiota dysbiosis, oxidative stress, lipid metabolic disturbances, and increased cardiovascular burden (13, 14). These findings raise the possibility that, although functional foods and performance-oriented beverages provide nutritional benefits to athletes and fitness enthusiasts, the inclusion of non-dairy creamer may diminish or counteract these advantages (15).

Despite the widespread use of FHVO-NDC in sports-related foods, the combined effects of this ingredient on muscle physiology, metabolomic profiles, and gut microbiota have not been examined in a controlled experimental model. To address this research gap, the present study proposes a hypothesis: intake of FHVO-NDC impairs muscle traits by altering intestinal microbiota composition and serum metabolite profiles, ultimately leading to reduced athletic performance (Figure 1).

Mice were selected as the experimental model because the core physiological and molecular systems underlying exercise capacity, such as muscle contraction mechanics, mitochondrial energy metabolism, and neuromuscular regulation, are highly conserved between mice and humans (16). Moreover, murine models allow precise control of diet, environment, and activity, enabling the identification of relationships that are difficult to isolate in human populations due to confounding factors (17). Therefore, mice represent an appropriate in vivo system for evaluating the physiological impacts of exogenous food additives, providing mechanistic insights that are relevant to human athletic performance (18, 19).

Given that athletic performance is closely influenced by body condition, feeding behavior, myofiber characteristics, biochemical fatigue markers, serum metabolites, and gut microbial composition, the objective of this study was to investigate how FHVO-NDC intake affects these parameters using a controlled mouse model. The anticipated findings will contribute to a deeper understanding of how FHVO-NDC consumption may influence exercise capacity and recovery, offering meaningful dietary implications for athletes, fitness enthusiasts, and the sports nutrition industry.

Fatty acid composition analysis of FHVO-NDC in the present study revealed that this product contained high levels of saturated fatty acids (37.44%), ranked as follows: lauric acid (14.26%), stearic acid (7.39%), myristic acid (5.37%), palmitic acid (4.42%), capric acid (2.67%), caprylic acid (2.25%), arachidic acid (0.96%), and behenic acid (0.12%). In contrast, unsaturated fatty acids were relatively less abundant, including oleic acid (3.28%), linoleic acid (0.34%), and eicosenoic acid (0.19%) (Table 1).

Changes in the body condition of mice following FHVO-NDC intake are shown in Table 2. No significant differences in body weight, body length, body surface area, or BMI were observed at any measurement timepoint.

Additionally, FHVO-NDC intake had no significant effects on appetite-related parameters, including daily feed intake and water consumption (Table 3).

Consumption of FHVO-NDC significantly reduced forelimb grip strength on day 42 (p = 0.001), 56 (p = 0.006), and 70 (p < 0.001) (Table 4). Moreover, mice in the TRT group exhibited poorer performance in the exhaustive swimming test (p = 0.030), wire hanging test (p = 0.012), and pole test (p = 0.011) (Table 4).

The relative weight of the gastrocnemius muscle was significantly reduced by FHVO-NDC administration (p = 0.021; Table 5). In addition, FHVO-NDC consumption resulted in decreased myofiber diameter (p < 0.001), reduced muscle glycogen storage (p < 0.001), and lower levels of key metabolic and ionic transport enzymes, including pyruvate kinase (p < 0.001), succinate dehydrogenase (p < 0.001), Na⁺/K⁺-ATPase (p < 0.001), and Ca²⁺-Mg²⁺-ATPase (p = 0.001) (Table 5).

Serum metabolite profiling revealed widespread metabolic alterations following FHVO-NDC intake. A total of 442 metabolites were significantly upregulated and 884 were downregulated (Figure 2A). Among these, 2-fluoronicotinic acid and PG (16:0/18:1) were identified as key upregulated metabolites based on their high rankings in the top 20 metabolites by log₂ fold change (Figure 2B). Comparison analysis further showed that levels of 2-fluoronicotinic acid (p = 0.022) and PG (16:0/18:1) (p = 0.040) were significantly higher in the TRT group than in the CON group (Table 6).

Regarding the intestinal microbiota, alpha diversity indexes, including Chao1, Simpson, Shannon, and Pielou did not differ significantly between groups (Table 7). Similarly, beta diversity analyses using Adonis, Amova, Anosim, and MRPP showed no statistically significant separation among microbial communities (Table 8).

LEfSe analysis identified several taxa that differed significantly between groups. Alistipes, ASF356 (Clostridium), and Escherichia_Shigella were enriched in mice consuming FHVO-NDC, whereas Lactobacillus was the signature genus in the CON group (Figures 3A,B). Random forest analysis further evaluated the relative importance of these taxa in relation to muscle-related phenotypes. Both the mean decrease accuracy and mean decrease Gini indicators ranked Alistipes as the most influential genus. Comparison analysis confirmed that Alistipes abundance was significantly higher in the TRT group than in the CON group (p = 0.007; Table 6).

Furthermore, Spearman correlation analysis revealed that the abundance of Alistipes was positively correlated with serum 2-fluoronicotinic acid levels (p = 0.029; Table 6).

Body condition and feeding behavior are critical parameters for evaluating the general health status of experimental animals, as they reflect both physiological stability and overall adaptability to experimental conditions. Under adverse or stressful conditions, animals often display poorer body condition, abnormal feeding patterns, or reduced food intake, which may indicate discomfort or systemic dysfunction (51). Ali et al. (52) reported that the body weight of mice consuming non-dairy creamer remained comparable to that of mice consuming milk, suggesting that the intake of such products does not necessarily disturb basic physiological functions. Consistent with these findings, the present study showed that consumption of water containing FHVO-NDC did not affect any parameters related to body condition or feeding behavior. These observations collectively suggest that FHVO-NDC intake did not induce acute physical discomfort in mice and that any subsequent physiological or metabolic changes identified in this study are unlikely to be attributed to poor feeding behavior or compromised body condition.

From the perspective of its fatty acid composition, saturated fatty acids are the primary components of FHVO-NDC. High intake of saturated fatty acids has been shown to increase the risk of metabolic syndrome (7, 8) and to promote chronic low-grade inflammation (9). Both metabolic syndrome and chronic inflammation are recognized risk factors for impaired musculoskeletal health, and they have been associated with reductions in muscle mass and strength (10, 11). Moreover, chronic inflammation can directly disrupt muscle metabolism, impair neuromuscular function, and ultimately reduce athletic performance (12). Therefore, strategies that prevent metabolic syndrome or suppress chronic inflammation have been shown to effectively improve physical performance and muscle function (53, 54). Although one of the saturated fatty acids in FHVO-NDC, lauric acid, has been reported to support myofiber formation and promote skeletal muscle growth (55, 56), other major saturated fatty acids present in FHVO-NDC exert detrimental effects. For example, stearic acid promotes proinflammatory cytokine release (57), contributes to cell growth inhibition, and induces cellular inflammation (58). Myristic acid contributes to adipose tissue inflammation (59) and has been linked to hypercholesterolemia (60). Palmitic acid, the most widely studied saturated fatty acid, is known to trigger metabolic inflammation, mitochondrial fragmentation, and reductions in mitochondrial membrane potential, all of which may impair exercise capacity and energy production (61, 62). Additionally, myristic acid has been shown to aggravate palmitic acid-induced inflammation (63), suggesting a synergistic inflammatory effect when multiple saturated fatty acids coexist. Taken together, these findings suggest that although FHVO-NDC contains some beneficial components, its overall saturated fatty acids components may contribute to reduced athletic performance.

As expected, throughout the experiment, mice in the TRT group showed insufficient strength to complete the swimming, wire-hanging, and pole-climbing tasks for extended periods. Athletic performance is closely linked to skeletal muscle integrity, particularly muscle mass and myofiber diameter (64, 65). Histologically, skeletal muscle is composed of long, cylindrical myofibers arranged in bundles, and these myofibers serve as the fundamental functional units of muscle tissue (66). Numerous studies have shown that myofiber diameter is directly proportional to muscle strength and total muscle mass (67–69). Conversely, a reduction in myofiber diameter is considered a hallmark histopathological feature of skeletal muscle atrophy (70). Therefore, larger myofibers and greater muscle mass generally correspond to enhanced athletic capacity (71, 72). In the present study, the significant reductions observed in gastrocnemius muscle mass and myofiber diameter in the TRT group suggest that FHVO-NDC intake may induce muscle atrophy in mice, which is consistent with their impaired athletic performance.

Furthermore, muscle glycogen content, a major energy reserve within muscle tissue, is typically positively correlated with muscle mass. Glycogen represents the primary storage form of carbohydrates in animals (73). During exercise, glycogen is degraded into glucose, which subsequently undergoes metabolic processes to generate ATP (74). Larger muscles generally possess a greater capacity for glycogen storage, allowing for more sustained energy output during physical activity (75). Insufficient glycogen storage leads to rapid fatigue and impaired athletic performance (76), and several studies have confirmed that interventions promoting glycogen synthesis enhance exercise capacity (77). Therefore, the reductions in glycogen storage capacity may also contribute to the decline in athletic performance. In addition to glycogen, muscle energy metabolism depends on the activity of several key enzymes involved in ATP production. We evaluated the levels of pyruvate kinase, succinate dehydrogenase, Na⁺/K⁺-ATPase, and Ca²⁺-Mg²⁺-ATPase, enzymes essential for glycolysis, the tricarboxylic acid cycle, and ion transport-dependent ATP consumption (78, 79). Reduced levels of these enzymes has been linked to impaired muscle energy metabolism and decreased exercise capacity, whereas higher levels support improved muscle function and endurance (78, 79). In this study, FHVO-NDC intake decreased all four ATP production-related enzymes, indicating substantial disruption of muscle energy metabolism. Therefore, the combined reduction in ATP-producing enzymes and lower glycogen concentrations directly compromises the energy supply available for muscle contraction, providing a mechanistic explanation for the observed decline in athletic performance.

Circulating metabolites can regulate the levels of enzymes involved in cellular energy production through multiple biochemical mechanisms (80). In the present study, 2-fluoronicotinic acid and PG(16:0/18:1) were identified as the two key upregulated metabolites, as both were ranked within the top 20 metabolites based on log₂ fold change values. PG(16:0/18:1), a phosphatidylglycerol species, has been previously associated with reductions in skeletal muscle mass and functional capacity (81). Consistently, Hinkley et al. (81) demonstrated that elevated phosphatidylglycerol levels are characteristic of sarcopenic muscle and show a negative correlation with muscle volume, suggesting a potential role in muscle wasting. In addition, 2-fluoronicotinic acid has been reported to inhibit nicotinate phosphoribosyltransferase (NaPRTase) activity in human platelet lysate (82). NaPRTase catalyzes the synthesis of nicotinic acid mononucleotide, the direct precursor required for the formation of nicotinamide adenine dinucleotide (83). As nicotinamide adenine dinucleotide availability is closely linked to efficient ATP generation through oxidative metabolism (84), suppression of this pathway may impair cellular energy production. Therefore, the elevated levels of PG (16:0/18:1) and 2-fluoronicotinic acid observed in this study likely contribute to the reduced muscle strength and diminished ATP production capacity.

Circulating metabolites are strongly influenced by the intestinal microbiota (85). In this study, we first evaluated the overall structural characteristics of the intestinal microbiota by assessing alpha diversity indices, including Chao1, Simpson, Shannon, and Pielou, which reflect microbial richness, diversity, stability, and evenness, respectively (86). To further examine compositional differences among groups, beta diversity was analyzed using multiple statistical approaches, including Adonis, Amova, Anosim, and MRPP, thereby providing a robust assessment of community structural variation. Results from both alpha diversity and beta diversity analyses showed no significant differences among groups, suggesting that FHVO-NDC intake did not alter the richness, diversity, stability, or evenness of the gut microbial community. However, it is important to note that unchanged alpha diversity and beta diversity metrics do not necessarily reflect functional or taxonomic stability. Microbial communities can maintain comparable richness and overall evenness while undergoing taxonomic turnover or shifts in specific bacterial populations (86, 87). Therefore, despite the absence of diversity changes, FHVO-NDC consumption may still influence particular microbial taxa that contribute to metabolic alterations and the observed decline in athletic performance.

To further investigate microbial differences, we employed the LEfSe algorithm to identify signature taxa within the intestinal microbiota. The analysis revealed that Alistipes, ASF356 (Clostridium), and Escherichia_Shigella were signature genera enriched in mice consuming FHVO-NDC, whereas Lactobacillus was identified as the key genus in the CON group. The Lactobacillus genus is widely recognized as beneficial and has been associated with improved muscle function, metabolic stability, and enhanced exercise capacity (88). In contrast, Clostridium, Alistipes, and Escherichia_Shigella are generally regarded as harmful bacteria that have been linked to inflammation, metabolic dysfunction, and impaired muscle performance (89–91). These findings suggest that FHVO-NDC intake may shift the intestinal microbiota toward a more deleterious composition despite the absence of changes in overall microbial diversity.

To further evaluate the relative contributions of these genera, we applied a random forest ensemble learning method, which provides an importance ranking based on mean decrease accuracy and mean decrease Gini. Both indicators consistently ranked Alistipes as the most influential genus. This observation strongly supports the notion that the expansion of Alistipes plays a central role in mediating the negative effects of FHVO-NDC on muscle function and athletic performance. Similar conclusions were reached by Lee et al. (90), who identified Alistipes as a predominant genus in sarcopenic and aged mice. Furthermore, Kim et al. (92) reported a positive correlation between Alistipes abundance and chronic fatigue syndrome. Together, these studies reinforce the concept that Alistipes is closely associated with muscle weakness phenotypes. Mechanistically, Alistipes possesses the genes required for fatty acid degradation and can efficiently utilize dietary fatty acids as a growth substrate (93). Given the saturated fatty acid-rich composition of FHVO-NDC, the increased abundance of Alistipes in the TRT group is therefore biologically plausible. Moreover, Spearman’s correlation analysis further demonstrated that 2-fluoronicotinic acid was positively correlated with Alistipes abundance, suggesting a potential interaction between circulating metabolite production and microbial community shifts. This correlation indicates that increases in Alistipes may contribute to metabolic alterations.

Therefore, this study provides an integrated assessment of the effects of FHVO-NDC intake on athletic performance by combining behavioral tests, muscle histology, biochemical analysis, metabolomics, and gut microbiota profiling. The identification of key metabolites [2-fluoronicotinic acid and PG(16:0/18:1)] and harmful bacteria such as Alistipes offers new mechanistic insights into how hydrogenated fat-based creamers may impair muscle function.

However, several limitations should be noted. First, the study was conducted only in mice, and their physiological responses may not fully represent humans. Second, although associations between metabolites, microbes, and muscle performance were observed, causal relationships were not directly validated using functional experiments. Third, only one dose and duration were tested; dose–response or time-dependent effects remain unknown. Fourth, the relatively small sample size may reduce statistical power. In addition, underlying molecular pathways such as mitochondrial function and muscle-fiber remodeling require further clarification.

Despite these limitations, the findings suggest that FHVO-NDC intake may negatively affect muscle traits and athletic performance by altering serum metabolism, reducing glycogen storage, decreasing ATP-producing enzymes, and increasing harmful gut bacteria. These results raise concerns about frequent consumption of hydrogenated fat-based creamers. Although direct dietary recommendations for athletes cannot yet be made, limiting excessive intake may be prudent.

Future studies should include dose–response experiments, sex-difference analyses, and human clinical validation. Mechanistic research on mitochondrial bioenergetics and metabolite-microbiota interactions will also help translate these findings into evidence-based dietary guidance for physically active individuals.

Based on the findings of this study, although no changes in body condition or feeding behavior were observed, the consumption of FHVO-NDC resulted in significant negative effects on athletic performance of mice. This decline is closely associated with decreased muscle glycogen storage and reduced levels of enzymes related to muscle fatigue, driven by reductions in muscle mass and myofiber diameters, both of which are crucial for energy production during exercise. Furthermore, serum metabolomics and intestinal microbiota analyses revealed that FHVO-NDC intake promoted the accumulation of metabolites, such as PG (16:0/18:1) and 2-fluoronicotinic acid, which are associated with impaired muscle function and reduced ATP production. Alterations in the intestinal microbiota, particularly the increase in Alistipes, Escherichia_Shigella, and Clostridium, further contributed to the observed decline in athletic performance.

Therefore, our findings provide preliminary evidence that hydrogenated fat-based creamers could detrimentally alter metabolic and microbiome profiles relevant to exercise performance, and may further negatively affect muscle traits, leading to reduced glycogen stores and decreased levels of ATP-producing enzymes, ultimately compromising athletic performance of mice.