Influence of Intermittent Fasting on Body Composition, Physical Performance, and the Orexinergic System in Postmenopausal Women: A Pilot Study

Postmenopausal women experience significant physiological changes, including increased visceral fat and decreased muscle mass, primarily due to hormonal shifts, particularly the decline in estrogen. These changes promote systemic inflammation, insulin resistance, and a higher risk of metabolic syndrome, negatively impacting physical function, cardiovascular health, and overall quality of life [1]. Addressing these metabolic disturbances is essential for improving health outcomes in this population, and targeted strategies combining nutrition and exercise have gained increasing attention.

Intermittent fasting has emerged as a promising approach for optimizing body composition and metabolic health. This strategy alternates periods of eating and fasting with popular methods such as the 16:8 protocol (sixteen hours of fasting and an 8-h eating window) and alternate-day fasting [2,3]. Research shows that IF enhances metabolic flexibility and the body’s ability to switch between glucose and fat as primary fuel sources by promoting fat oxidation, improving insulin sensitivity, and reducing inflammation [4,5,6]. These effects are driven by cellular responses to energy deficits, including activating AMP-activated protein kinase (AMPK) and autophagy, improving glucose regulation, mitochondrial function, and oxidative stress resistance [7].

Beyond these metabolic benefits, IF profoundly affects the neuroendocrine system, particularly the orexinergic system. Orexin-A (hypocretin-1), a neuropeptide produced in the lateral hypothalamus, plays a key role in regulating energy balance, arousal, and feeding behavior by activating its receptors, OX1R and OX2R [8]. When energy availability decreases, such as during fasting, orexin neurons respond by stimulating neuropeptide Y (NPY) and agouti-related peptide (AgRP), increasing appetite to restore energy balance while simultaneously activating the sympathetic nervous system (SNS) to boost thermogenesis and lipolysis [9,10,11]. This dynamic coordination helps the body balance energy intake and expenditure in response to metabolic stress.

Importantly, AMPK, a master regulator of cellular energy, activates orexin neurons during fasting, driving lipid metabolism and glucose uptake while promoting mitochondrial biogenesis via peroxisome proliferator-activated receptor γ coactivator 1-alpha [12]. These processes enhance metabolic flexibility, efficiently helping the body transition between fuel sources. The potential for improving mitochondrial function and fat oxidation is directly relevant for postmenopausal women, as it can counteract the metabolic slowdown and visceral fat accumulation associated with menopause [13].

Pairing IF with HIIT may amplify these neuroendocrine and metabolic responses. High-intensity interval training consists of short bursts of intense exercise followed by brief recovery periods, a format known to enhance insulin sensitivity, fat oxidation, and cardiorespiratory fitness [14]. Recent studies suggest that HIIT activates orexinergic pathways by inducing metabolic stress, increasing AMPK activation, and stimulating lipid metabolism and muscle glucose uptake [15,16].

Crucially, fasting and HIIT work synergistically by engaging overlapping metabolic and neuroendocrine pathways. Intermittent fasting promotes metabolic switching, shifting from glucose to fat as the primary fuel, while HIIT triggers acute stress responses via the hypothalamic–pituitary–adrenal (HPA) axis, stimulating cortisol release and orexin activity to support energy mobilization [17]. This dual activation may enhance OX-A levels more effectively than either intervention alone, promoting better energy homeostasis and greater flexibility in fuel utilization. These physiological changes directly translate to practical outcomes for postmenopausal women. Enhanced orexinergic activity supports fat metabolism and muscle function and influences motivation for physical activity by activating the mesolimbic dopamine system, the brain’s reward pathway [18,19]. This link between orexin and motivation suggests that combining IF with HIIT could help improve metabolic health and exercise adherence, an often-overlooked factor in long-term weight management.

Despite these promising connections, the interaction between IF, HIIT, and the orexinergic system remains underexplored, especially in postmenopausal women, whose neuroendocrine activity may already be altered by aging [20]. Given that OX-A plays a pivotal role in regulating energy balance, stress responses, and physical performance, understanding how these interventions modulate orexin activity could open new avenues for tailored strategies to combat metabolic dysfunction and visceral fat accumulation in this population [13].

This study investigates the combined effects of IF and LCD with HIIT on body composition, physical performance, and OX-A levels in postmenopausal women. By exploring the neuroendocrine mechanisms underlying these interventions, the objective is to identify how dietary and exercise strategies influence orexinergic activity and improve metabolic health. This research could inform personalized approaches to enhancing energy regulation, reducing visceral fat, and supporting physical resilience in aging populations.

The primary aim of this study was to examine the combined effects of IF and LCD, integrated with a HIIT intervention, on body composition, physical performance, and OX-A levels in postmenopausal women. Analysis of the collected data (see Table 1) indicated that at T0, the group following the LCD diet covered a greater average distance (553.33 m) compared with the IF group (480.17 m). However, by T1, the IF group demonstrated a significant increase in performance, achieving an average distance of 610.08 m, which surpassed the 603.50 m covered by the LCD group. This suggests a positive response from the IF group to the combined diet and exercise intervention. In addition, OX-A levels exhibited an intriguing trend. The IF group started with a higher level (53.26 pg/mL) compared with the LCD group (35.18 pg/mL), and these values soared at T1, reaching 143.89 pg/mL for the IF group, while the LCD group increased to only 63.68 pg/mL. This indicates significant metabolic regulation in response to dietary and exercise changes. Furthermore, maximal oxygen uptake and average HR also experienced notable improvements. Specifically, VO2 max increased more in the IF group, highlighting the effectiveness of combining HIIT with IF in enhancing aerobic capacity.

An ANOVA (see Table 2) indicated statistically significant differences between groups at T0 and T1.

At T0, significant variations were observed in the 6MWT distance (F = 4.818, p = 0.039), mean HR (F = 5.943, p = 0.023), and sit-and-reach test (F = 15.849, p = 0.001), suggesting baseline differences in flexibility, cardiovascular response, endurance, perceived exertion, and metabolic regulation. At T1, OX-A levels (F = 10.931, p = 0.003) remained significantly different between groups, indicating continued differences in perceived exertion and metabolic regulation. Significant changes were also observed in 6MWT distance (F = 4.998, p = 0.036), VO2 max (F = 5.03, p = 0.035), and resistance (F = 6.73, p = 0.017), reflecting alterations in body composition, functional exercise capacity, and aerobic capacity.

Key predictors of post-intervention OX-A levels were identified (see Figure 2 and Figure 3). At T0, sit-and-reach test scores and baseline OX-A levels were the strongest positive predictors. At T1, VO2 max was a significant negative predictor, suggesting an inverse relationship between aerobic capacity and OX-A levels. The ranking of predictor importance was sit-and-reach test (T0), OX-A (T0), sit-and-reach test (T1), VO2 max (T1), and TAV (T1).

A scatterplot (see Figure 4) showed a positive correlation between observed and predicted post-intervention OX-A levels, with some deviations, indicating that the regression model explained part of the variance. Regression analysis confirmed the significance of these predictors, with the sit-and-reach test and baseline OX-A levels positively predicting post-intervention OX-A, and VO2 max negatively predicting it.

Regression analysis further identified the key predictors influencing post-intervention OX-A levels. At T0, the sit-and-reach cm transformed emerged as the most significant positive predictor, closely followed by the baseline OX-A transformed. In contrast, at T1, VO2 max mL/g/min transformed negatively impacted OX-A levels, indicating a possible inverse relationship between aerobic capacity and orexinergic activity post-intervention. Table 3 provides a detailed analysis of the key predictors of post-intervention OX-A levels, highlighting the interconnected effects of flexibility, metabolic function, and aerobic fitness. Pearson correlation analysis (see Table 3) revealed significant associations between physiological and performance measures. T0 mean HR was negatively correlated with T1 OX-A levels (r = −0.573, p = 0.033), and T0 6MWT distance was positively correlated with the T0 mean HR (r = 0.611, p = 0.033).

Cohen’s d analyses (see Table 4) showed distinct effect sizes across dietary groups IF and LCD at T0 and T1. At T0, the LCD group showed superior endurance capacity (distance covered, d = −0.90) and flexibility (sit and reach, d = −1.63), while the IF group showed higher aerobic capacity (VO2 max, d = 0.36), perceived exertion tolerance (RPE, d = 0.77), and OX-A levels (d = 0.73). At T1, the IF group showed greater improvements in RPE (d = 1.72), VO2 max (d = 0.92), and sit-and-reach (d = 0.32), while the LCD group showed better adaptations in reactance (d = −1.46). The most significant shift was in sit-and-reach performance for the IF group (Δd = 1.94). Orexin-A levels showed a moderate positive shift for the IF group (Δd = 0.62).

These findings offer critical insights into how nutritional strategies and exercise interventions influence physical performance and metabolic biomarkers in postmenopausal women, emphasizing the role of flexibility and initial OX-A levels as key predictors in understanding these adaptations. At T0, Cohen’s d values indicated differing advantages between the IF and LCD groups across various metrics. A negative effect size for distance covered (d = −0.90) favored the LCD, suggesting superior baseline endurance capacity in this group. Conversely,max (d = 0.36) and RPE (d = 0.77) showed a performance advantage for the IF group, indicating higher initial aerobic capacity and perceived exertion tolerance. The sit-and-reach test exhibited a substantial adverse effect size (d = −1.63), implying greater baseline flexibility in the LCD group. In contrast, OX-A levels showed a moderate positive effect size (d = 0.73), favoring the IF group, suggesting a potential metabolic advantage in orexinergic activity at the study’s onset. Following the intervention, Cohen’s d analyses revealed significant shifts, particularly favoring the IF group in key physiological measures. Rate-perceived exertion (d = 1.72) andmax (d = 0.92) demonstrated substantial improvements in the IF group, reflecting enhanced aerobic fitness and exercise tolerance. Although the sit-and-reach test effect size shifted positively (d = 0.32), the change was minor, indicating a less pronounced improvement in flexibility over time. The most striking shift in Cohen’s d values was observed in post-intervention sit-and-reach performance (Δd = 1.94), highlighting a significant improvement in flexibility for the IF group. Conversely, reactance values (Δd = −1.46) shifted toward the LCD, suggesting better adaptations in bioimpedance measures for this group. Additionally, OX-A levels exhibited a moderate positive shift (Δd = 0.62), reinforcing the increasing metabolic effect of the IF diet over time. VO 2 VO 2

This study examined the physiological and neuroendocrine effects of IF and LCD in postmenopausal women, highlighting their impact on body composition, physical performance, and OX-A dynamics. By integrating a HIIT program, this study explores how dietary strategies and exercise interventions modulate metabolic and hormonal responses, offering more profound insights into the orexinergic system’s role in energy balance, cardiovascular function, and physical performance.

Our results show that both IF and LCD produced distinct metabolic benefits, but the addition of HIIT appeared to amplify neuroendocrine adaptations. A significant increase in post-intervention OX-A levels was observed, particularly in the IF + HIIT group. Since OX-A is a key neuropeptide regulating energy homeostasis, arousal, and feeding behavior, this rise suggests a direct link between intense physical activity, fasting states, and enhanced orexinergic activity. The observed increase in OX-A likely reflects the combined effects of intermittent metabolic switching, induced by IF, and acute stress responses (triggered by HIIT), a finding consistent with Messina et al. [11], who emphasized orexin’s role in promoting fat oxidation and insulin sensitivity. Orexin neurons in the hypothalamus are highly sensitive to energy deficits, becoming more active during fasting and exercise to stimulate lipid metabolism and glucose uptake via the AMPK pathway [11]. This aligns with existing research indicating that IF activates the “metabolic switch,” shifting the body from glucose to lipid metabolism, which enhances fat oxidation and improves metabolic efficiency [30].

Furthermore, IF has been shown to reduce oxidative stress and inflammation, likely mediated by metabolic switching and the upregulation of cellular repair mechanisms [1]. The fat mass reduction observed in the IF group mirrors findings by Varady et al. [4], who reported significant fat loss following alternate-day fasting.

The LCD group also demonstrated substantial reductions in fat mass and improvements in metabolic markers, consistent with Stockman et al.’s research [6]. The LCD’s effect appears tied to caloric restriction and the metabolic adaptation of using ketone bodies as an alternative energy source, potentially contributing to the anti-inflammatory responses noted in previous studies [31]. However, LCD may have a differential long-term impact compared with IF. The sustained caloric deficit inherent in LCD can lower energy expenditure over time, a known metabolic adaptation that may explain the weight-loss plateau some participants experienced, aligning with observations by Harris et al. [7].

Our study also shed light on the orexinergic system’s broader role in metabolic regulation. Beyond its impact on fat metabolism and glucose uptake, orexin modulates the HPA axis, a crucial stress response pathway. The activation of orexin neurons during HIIT likely stimulated cortisol release, which can drive lipid mobilization and energy expenditure [18]. This dual activation of orexin and HPA pathways suggests that the IF + HIIT protocol triggers a hormonal stress response: repeated, low-level stressors, like fasting and intense exercise, provoke adaptive neuroendocrine changes that enhance metabolic flexibility and resilience [32].

A novel finding of this study was the link between baseline sit-and-reach performance and post-intervention OX-A levels. Our regression analysis showed that greater flexibility positively predicted OX-A responses, a relationship that may stem from the orexin system’s role in muscle glucose uptake and energy regulation. This association echoes research by Kim et al. [33], suggesting that musculoskeletal health can influence metabolic outcomes. These findings broaden the view of physical fitness, indicating that beyond strength and endurance, flexibility may also shape hormonal responses and metabolic efficiency.

Interestingly, VO2 max at the end-point negatively predicted OX-A levels, pointing to a complex interaction between aerobic capacity and orexinergic activity. While HIIT improved cardiorespiratory fitness, the inverse relationship with OX-A may reflect a neuroendocrine balancing act: as aerobic capacity rises, the body may rely less on orexin signaling, shifting towards other metabolic regulators like catecholamines and glucocorticoids [20]. This nuanced relationship highlights the need for further research into how fitness markers interact with neuroendocrine pathways.

Our correlation analysis revealed significant links between cardiovascular fitness, physical performance, and OX-A levels. The negative correlation between baseline HR and post-intervention OX-A suggests that participants with lower initial cardiovascular fitness experienced greater orexinergic activation after IF and HIIT. This may indicate a compensatory response, where individuals with lower fitness rely more heavily on orexin neurons to balance energy demands during fasting and intense exercise. The positive correlation between distance covered and baseline HR further supports the idea that HR dynamics mirror neuroendocrine activity, especially when HIIT challenges the body to regulate energy expenditure and stress responses.

Cohen’s d analysis offered valuable insights into the practical significance of these neuroendocrine changes. At baseline, the IF group exhibited highermax and perceived exertion, suggesting a greater reliance on orexin-driven energy pathways. Post-intervention, the IF + HIIT group showed dramatic improvements in RPE andmax, reinforcing the idea that fasting and high-intensity exercise bolster aerobic capacity and neuroendocrine resilience. VO 2 VO 2

The most striking change was seen in sit-and-reach performance, supporting the emerging link between flexibility and metabolic health, a relationship potentially mediated by the orexinergic system’s regulation of muscle glucose uptake [34].

These findings are particularly relevant for postmenopausal women, a population often facing hormonal imbalances, visceral fat accumulation, and reduced neuroendocrine activity. The combination of IF and HIIT offers a dual-action strategy, enhancing metabolic flexibility while stimulating the orexinergic system to mitigate the metabolic decline associated with menopause [33]. By improving insulin sensitivity, reducing inflammation, and boosting cardiovascular health, these strategies may help manage obesity-related conditions prevalent in this demographic. Ultimately, this study highlights the neuroendocrine benefits of pairing IF with HIIT, demonstrating how these interventions work together to regulate energy homeostasis, metabolic function, and physical performance through orexin-mediated pathways.

As OX-A continues to emerge as a crucial player in metabolic and hormonal health, future research should investigate how diet and exercise strategies can further optimize neuroendocrine responses, unlocking new ways to promote metabolic health and longevity in aging populations.

While this study highlights the physiological and neuroendocrine benefits of combining IF, LCD, and HIIT in postmenopausal women, several limitations should be considered. First, the short intervention period may not fully capture the long-term neuroendocrine adaptations or the sustainability of these dietary and exercise strategies. While notable increases in OX-A levels suggested heightened energy regulation and metabolic flexibility, it remains unclear whether these effects persist over extended periods or if the body adapts to prolonged fasting and high-intensity exercise in ways that may alter orexinergic activity. Future studies should adopt longitudinal designs to explore how these interventions impact hormonal and metabolic pathways over time.

Second, although this study focused exclusively on postmenopausal women, gender differences in neuroendocrine responses to both diet and exercise warrant further investigation. Research shows that hormonal fluctuations, such as those involving estrogen and cortisol, can modulate orexin signaling and energy homeostasis [20]. Understanding these differences is crucial, as men and women may exhibit varying metabolic and neurohormonal adaptations to HIIT and fasting protocols.

Moreover, the healthy cohort used in this study may limit the generalizability of our findings to metabolically challenged populations, such as individuals with type 2 diabetes, obesity, or metabolic syndrome, who may experience dysregulated orexin pathways. Given the orexinergic system’s role in insulin sensitivity, fat oxidation, and energy balance, future research should investigate how these interventions affect those with impaired metabolic function.

Another limitation lies in the psychological factors influencing dietary adherence and exercise performance. While OX-A is known to modulate reward pathways and cravings, it did not directly assess how hunger, satiety, or mood fluctuations impacted participant behavior. Since HIIT and fasting may trigger both physiological stress responses via the HPA axis and psychological stress due to caloric restriction, future studies should integrate behavioral measures to better understand the neuropsychological dimensions of these interventions.

Finally, although the synergistic effects of HIIT and IF are acknowledged, which appear to amplify orexinergic activity and enhance metabolic regulation, it did not empirically isolate the effects of exercise alone versus diet alone. This leaves a gap in understanding how each component contributes independently and interactively to neuroendocrine outcomes. Future research should employ multifactorial designs to dissect the specific and combined roles of HIIT, fasting, and caloric restriction, tailoring interventions to individuals’ metabolic profiles and fitness levels. Ultimately, addressing these limitations will pave the way for personalized strategies that harness the neuroendocrine benefits of diet-exercise combinations, offering targeted solutions for improving metabolic health, energy regulation, and physical performance in both clinical and healthy populations.

This study highlights the interconnected metabolic, physiological, and neuroendocrine benefits of IF and LCD in postmenopausal women, emphasizing their effects on body composition, physical performance, and OX-A regulation. Both dietary strategies contributed to fat mass reduction and improved metabolic health, with IF enhancing metabolic flexibility and fat oxidation and LCD supporting fat loss through sustained caloric restriction. Crucially, both interventions positively influenced OX-A levels, linking dietary strategies to energy regulation and metabolic efficiency. The combination of IF and HIIT emerged as a particularly effective approach, showing a synergistic effect on visceral fat reduction, aerobic capacity, and orexinergic activity. This suggests that pairing targeted dietary strategies with structured exercise programs not only optimizes fat metabolism but also activates neuroendocrine pathways, offering a powerful strategy for improving physical performance and metabolic resilience in postmenopausal women. Furthermore, Cohen’s d analysis reinforced the practical significance of these interventions, highlighting meaningful improvements in key physiological markers. These findings present a compelling and sustainable approach to the management of obesity-related conditions and metabolic dysfunction during menopause, offering targeted strategies to counteract the hormonal and metabolic changes that often accompany this stage of life. Looking ahead, future research should focus on long-term neuroendocrine adaptations to these interventions, exploring how orexin pathways interact with individual metabolic profiles and how psychological factors, such as appetite regulation and exercise motivation, influence adherence and outcomes. Unraveling these complex relationships will be crucial in developing personalized diet-exercise programs that support metabolic health and functional performance in aging populations.

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/nu17071121/s1↗.

Conceptualization, A.A.V. and G.C.; methodology, P.V.; formal analysis, R.P., G.D. and R.R.R.M.; resources, G.C.; data curation, P.V. and M.T.; writing—original draft preparation, A.A.V. and P.V.; writing—review and editing, G.C.; supervision, G.M.; funding acquisition, G.C. All authors have read and agreed to the published version of the manuscript.

Ethics Committee of the Policlinico di Foggia (prot, n°440/15—EBTCDI) approved 15 November 2017.

Informed consent was obtained from all subjects involved in the study.

The original contributions presented in the study are included in the article and, further inquiries can be directed to the corresponding author. Supplementary Materials

The authors declare no conflicts of interest.

This research was funded by University of Foggia Deparment of Clinical and Experimental Medicine, Via Luigi Pinto-c/o Centro di Ricerche Biomediche “E. Altomare”, 71122 Foggia, Italy. VAT IT 03016180717.