Osteoporosis, a chronic bone metabolic disease characterized by weakened bones, which increases the risk of fractures, is a serious public health problem affecting millions of people, especially the elderly and postmenopausal women (Al-Ansari et al., 2022; Jaul and Barron, 2017). A bone fracture seriously affects the daily activities and quality of life of individuals and can result in complete paralysis or death (Al-Ansari et al., 2022). Worldwide, osteoporotic fracture occurs every 3 seconds, with over 8.9 million fractures occurring yearly (Jaul and Barron, 2017). The possible outcomes of vertebral compression fractures include pain, deformity, disability, and increased mortality (Al-Saleh et al., 2023). Furthermore, those with hip fractures experienced a significant increase in morbidity and medical expenses (Al-Saleh et al., 2023). Osteoporosis has many risk factors, such as disruptive endocrine changes like menopause, obesity, and vitamin D deficiency (Al-Ansari et al., 2022). Estrogen hormone deficiency may also lead to bone and muscle loss and impaired physical function, which leads to obesity, hyperleptinemia, and leptin resistance (Ezzat-Zadeh et al., 2017; Colleluori et al., 2022).

The prevalence of osteoporosis is noteworthy, even in regions like Saudi Arabia, where an epidemiologic analysis revealed that 34% of healthy women and 30.7% of men aged 50–79 years are osteoporotic (Ezzat-Zadeh et al., 2017). Given these statistics, screening recommendations suggest that women aged 65 years and older, or those between 60 and 64 years with additional risk factors, should undergo osteoporosis screening (Al-Ansari et al., 2022; Al-Saleh et al., 2023). Hence, early detection and intervention are crucial in mitigating the personal and societal costs associated with this condition.

A change in a lifestyle, such as quitting smoking, exercising, and following an appropriate diet, is one way to prevent osteoporosis (Alwahhabi, 2015). Exercise is considered to positively impact osteoporosis and bone mineral density (BMD) (Zhang et al., 2022). In this context, different types of physical exercise, including aerobic exercise, which speeds up the heart rate and breathing, strength exercise, balance exercise, high-impact exercise, resistance training, and plyometric exercise, which utilizes the stretch‐shortening cycle by using a lengthening movement (eccentric) which is quickly followed by a shortening movement (concentric), are used in clinical practice to maintain or increase BMD (Li et al., 2021). A study has reported that exercise can increase bone strength and functional performance in postmenopausal women (Watson et al., 2018). Recently, a study demonstrated an indirect effect of exercise on bone tissue, by reducing leptin levels and improving insulin sensitivity (Khalafi et al., 2023). Therefore, scientists began to study alternative diets such as fasting, which are thought to decrease the incidence of osteoporosis.

Intermittent fasting can increase bone mass by decreasing parathyroid hormones and osteoclastogenesis and enhancing the osteoblast mechanism in a rat model (Alrowaili et al., 2021). Ramadan fasting is a special type of fasting, where during fasting hours, eating, drinking, and smoking are not allowed, but individuals are allowed to eat or drink from sundown until sunset (Wang and Wu, 2022). Ramadan fasting hours range from 13 to 18 depending on the geographical area, as the fasting hours are more than intermittent fasting (Correia et al., 2020). The Ramadan model (RM) protects the body from pro-inflammatory factors including TNF-α and IL-6 (Correia et al., 2020). Moreover, a study found a significant reduction of lowering blood sugar and LDL after 1 month of RM (Jahrami et al., 2022). Several studies have suggested that there is an association between inflammatory factors and insulin resistance with increased bone resorption and, thus, osteoporosis (Sanguineti et al., 2014; Xia et al., 2012).

To date, the mechanisms behind how Ramadan fasting and exercise individually and in combination affect bone health in ovariectomized rats are not fully understood. Most previous studies have focused on either Ramadan fasting or exercise alone, and there is a lack of research examining the combined effects of Ramadan fasting and exercise on bone health. The primary objective of the current study is to observe the preventive approach of an exercise program combined with RFM on osteoporosis in ovariectomized rats. The aim of the study was to examine the preventive approach of Ramadan fasting model and exercise, individually and in combination, to determine the bone health on ovariectomy-induced osteoporosis in a rat model.

Osteoporosis is a worldwide common disease that leads to many complications and increases morbidity and mortality, which causes a burden on health-care systems (Al-Ansari et al., 2022; Jaul and Barron, 2017). Lifestyle modification is the first line of treatment, and few studies investigated the effect of lifestyle on preventing osteoporosis. The ovariectomized rat model is one of the approved animal models to mimic postmenopausal, induced osteoporosis (Li et al., 2014; Yousefzadeh et al., 2020). In this sense, the current study investigated the protective effect of exercises and RFM and their combination against osteoporosis on the OVX rat model. Our results showed significantly increased body weight among the OVX group compared with the control group. These findings are explained by an observational cohort study; osteoporosis in postmenopausal women may be due to their altered hormonal status, including estrogen and follicle-stimulating hormone (FSH), which are noted to be associated with obesity occurring in women (Ebong et al., 2022). Apparently, the drop in circulating estradiol and the increase in FSH went along with the shift in visceral adiposity. Furthermore, menopause is linked to an increase in visceral and total body fat due to estrogen’s effects on regulating the expansion and metabolism of adipocytes, including lipolysis and lipoprotein lipase activity, as well as the correlated relationship between FSH and lipid production (Ebong et al., 2022; Curtis et al., 2018).

According to Burch et al. (2021), ovariectomized rats experience estrogen level depletion, which consequently leads to a rapid and significant increase in body weight, whereas intact rats gain weight slowly and more moderately. Moreover, the OVX group shows a significant increase in free fatty acids (FFAS) and triglyceride levels from baseline to complete 8 weeks. At the end of the experiment period, it was observed that the OVX group gained more weight than the control group (Curtis et al., 2018). Another experimental study carried out by Damirchi et al. (2010) made an interesting observation that ovariectomy in rats leads to a dramatic increase in body weight, as shown in the study results by the eighth week. Furthermore, there was a higher visceral fat level in the OVX group than in the control rats. These results of the previous studies are consistent with the findings of the present study. As was foreseeable, ovariectomy was followed by a significant gain in body weight after 2 weeks of the experiment. At the seventh week, there was a significant difference in body weight gain between the OVX and control groups. Furthermore, the weight gain continued in the OVX group until the end of the trial at the eighth week. A study demonstrated the effectiveness of exercise in postmenopausal women by reducing 6%–7% of body weight, leading to positive consequences on estradiol, free estradiol, androstenedione, and free testosterone as well as improved physical fitness (Moro et al., 2016). An additional study discovered that exercise dramatically decreased visceral fat in the OVX, with the exercise group contrasted to the OVX group; however, the body weight and BMI remained unchanged (van Gemert et al., 2015). In addition to exercise, fasting improves the metabolic state and weight loss and changes in body composition. A study conducted for 8 weeks demonstrated the effectiveness of fasting for 16 h/8 h feeding concluded that there was a drop in fat mass (Agostini et al., 2018).

Consistent with these studies, our findings observed that there was a positive effect of fasting and exercise on body weight change over the study duration, as observed when comparing the OVX–RFM group and the OVX–Exercise group with the OVX group. While previous studies’ findings are instructive in the context of exercise and fasting effects on body weight, the current study provides the effect of exercise and fasting combined on the body weight. There was a huge difference in body weight gain in the OVX, OVX + Exercise, and OVX + RFM groups with the control groups at the seventh week. However, by the eighth week of the experiment, it was noted that the control group alongside the combination group (OVX + RFM + Exercise) had the lowest body weight gains in contrast with the OVX, OVX + Exercise, and OVX + RFM groups. Overall, post-menopause leads to hormonal chaining linked to obesity in women. Moreover, fasting and exercise help promote losing body weight. Thereby, the combination of fasting with exercise shows a positive effect in terms of losing weight in the OVX rats. Our results support the hypothesis in accordance with the previous study, and this study shows significant increase in body weight and significant decrease in bone markers among ovariectomized rats without intervention. Previous studies illustrated a relation between sarcopenia and osteoporosis due to the biomechanical and biochemical relationship between muscles and long bones’ tissues (Barengolts et al., 1993). Skeletal muscle produces many proteins and peptides that can impact bone health; therefore, exercise is beneficial for osteoporosis prevention and treatment (Barengolts et al., 1993).

A study suggests that in the early stages of estrogen deficiency, endurance exercise can postpone the initial stage of bone mineral loss (Bahijri et al., 2015) and promote the development of muscle mass, endurance, and strength. Physical activity demands additional strain, so the formation of bone occurs, whereas lower strains encourage bone resorption; in addition, exercise can increase the myokines and osteokines, which are secreted by the muscle and bone, respectively, and has anabolic effects on the muscle and the osteoblast lineage through improving bone-forming cell differentiation and activity (Barengolts et al., 1993). However, it has been demonstrated that resistance exercise has a higher impact than endurance exercise on bone mass maintenance and improvement because of the nature of the mechanical stimulus. In addition, if we perform an endurance exercise without the right caloric intake, we can have the opposite effect with the induced protein catabolism. Amato et al. (2022) demonstrated the efficacy of resistance training in the elderly population on bone remodeling.

Furthermore, through changes in bone structure and/or localized adaptation in bone distribution in the regions subjected to the greatest strain, exercise training may improve bone strength independent of changes in BMD (Allison et al., 2015; Warden et al., 2007). A bone’s resistance to bending increases during exercise as a result of increased cortical thickness brought on by load-induced periosteal apposition and, to a lesser extent, decreased endocortical resorption (Warden et al., 2014). In this regard, Krogh et al. (2024) recently found that resistance training led to significantly increased levels of fasting N-terminal propeptide of type-I procollagen (P1NP, a bone formation marker) in both pediatric hematopoietic stem cell transplantation patients and controls with no significant changes in fasting C-terminal telopeptide of type-I collagen (CTX, a bone resorption marker) levels.

Dietary adjustments related to fasting during Ramadan regulated PTH secretion in a way that would be advantageous to bone health (Adawi et al., 2017). Fasting may have a positive impact on bone turnover while also lowering pro-inflammatory markers. A shift in eating habits during RFM influences PTH secretion by raising the calcium content in the evening and reducing bone resorption at night (Adawi et al., 2017; Hisatomi and Kugino, 2019). Furthermore, the current study discovered that intermittent fasting for 16–18 h in rats led to a large rise in serum levels of osteoprotegerin and a significant drop in serum levels of RANK, indicating that RFM can suppress osteoclast activity and stimulate osteoblast activity (Park and Shin, 2022). Similarly, Alrowaili et al. (2021) found that in rats with glucocorticoid-induced osteoporosis that were subjected to intermittent fasting for 16–18 h per day for 90 days, the serum levels of the bone formation biomarkers, namely, osteoprotegerin, ALP, and osteocalcin, were significantly increased, and those of the bone resorption markers, namely, tartrate-resistant acid phosphatase (TRAP)-5b, amino-terminal cross-linking telopeptide of type-I collagen (NTX-1), and deoxypyridinoline (DPD), were significantly decreased. This suggests that intermittent fasting slows the progression of glucocorticoid-induced osteoporosis by inhibiting osteoclast activity and promoting osteoblast osteogenesis. In a similar vein, it was discovered that intermittent fasting counteracted both the rise in the serum bone resorption marker TRAP and the BMD reduction brought on by a ketogenic diet (Xu et al., 2019). Controversy around animal studies shows that fasting shows no significant differences in lumbar vertebral body height and cortical bone thickness compared to the non-fasting group; however, the duration of fasting in the study was only 4 days (Masanova et al., 2022).

Supporting the beneficial effect of fasting, in a 6-month randomized controlled trial, the effects of alternate-day fasting regimens and caloric restriction on bone metabolic markers in overweight and obese individuals were investigated in relation to religious Ramadan fasting. The results showed good weight reduction in both the caloric restriction and alternate-day fasting groups, and no significant effects on bone mineral content (BMC), bone mineral density (BMD), or the markers related to bone metabolism, type-I collagen carboxy-terminal peptide (CTX-1), and OPG (Barnosky et al., 2017) were observed. Comparable findings were reported by Clayton et al. (2020), who discovered no impact of a 24-h fast on serum levels of PTH, procollagen type-I N-terminal propeptide (PINP), or CTX-1. In addition, Martens et al. (2020) demonstrated that neither the total BMD nor the regional BMD of middle-aged and elderly non-obese individuals differed from a control group after 6 weeks of time-restricted feeding, nor did these individuals’ bone mass decrease.

In clinical studies, Ramadan fasting could relieve the symptoms of the patients with RA or SpA by regulating inflammatory cytokines (decreasing CRP, IL1, or 6), lipid profiles, antropometric features, or intestinal microbial compositions (Ben Nessib et al., 2021; Rodopaios et al., 2024). As RIF has been found to have a beneficial effect on the secretion of PTH, it was hypothesized that it could positively affect the bone metabolism. RIF has also a positive impact on the activation of dipeptidyl peptidase 4 (DPP-4) inhibitors as the DPP-4 gene has been identified as an important genetic factor contributing to the progression of osteoporosis (Ben Nessib et al., 2021; Bahijri et al., 2015). However, there are no clinical investigations and basic research that indicates that RIF and exercise can ameliorate osteoporosis.

Overall, there is a significant reduction in bone density among ovariectomized rats, especially in the concentrations of calcium and magnesium in the bones, and this is mainly due to the depletion in the estrogen level (Lei et al., 2009; Vaananen and Harkonen, 1996). Estrogen deficiency affects bone density by increasing PTH, IL-1β, and IL-6, which are cytokines that increase the process of osteoclasts formation (Stern et al., 1988; Masanova et al., 2022).

The current study results found that the femur bone calcium concentration and serum calcium concentrations were higher in the OVX + Exercise group than in the OVX group without interventions. Recently, in some studies, the relationship between calcium homeostasis and exercise had been investigated. Some experimental animal studies found that there is an increase in the absorption of calcium from the intestine due to the stimulation of exercise to increase bone density, which leads to an increase in vitamin D3 absorption, resulting in the inhibition of PTH secretion (Yeh and Aloia, 1990; Ashizawa et al., 1997). Furthermore, exercise can help balance calcium levels in the bones and blood (Tella and Gallagher, 2014). Calcium homeostasis is important because a chronical decrease in calcium serum concentrations is associated with increase in PTH levels, which leads to the replacement of calcium from the bones to the blood (Peterson and Heffernan, 2008).

The results of the current study also showed that there was a slight unremarkable elevation and hemostasis in the OVX + RFM group compared to the OVX group in the serum samples. To explain our results, a study by Park and Shin (2022) found that intermittent fasting protects bone mass by reducing TNF-α levels in the serum. TNF-α can affect calcium absorption by decreasing the level of calcitriol in the blood (Attarzadeh Hosseini et al., 2013). In contrast, previous studies that examined the effect of Ramadan fasting on blood calcium levels have shown that blood calcium levels may be normal or slightly low due to the increased excretion of calcium in the urine and due to PTH abnormalities after Ramadan fasting (Bahijri et al., 2015; Castiglioni et al., 2013).

The main results of the current study investigated the effect of exercise and fasting on OVX rats compared with the control group. There is an identical result of calcium concentration in the bones and serum of the OVX + RFM + Exercise group compared to that in the control group that had not undergone an ovariectomy surgery, and this may be due to the combined benefits of fasting and exercise in regulating PTH and RANKL levels.

In addition, magnesium deficiency affects the absorption of vitamin D and PTH, which affects the absorption of calcium and bone density (Vazquez-Lorente et al., 2020; Marcus, 2018). Furthermore Vazquez-Lorente et al. (2020) investigated the mechanism effect of magnesium in bones, and they found that hypomagnesemia was associated with a increase in cytokines that accelerate the process of osteoclast, resulting in an increase in bone stiffness and osteoclast’s function. In OVX rats, they were exposed to magnesium deficiency, as depletion of the estrogen level was associated with low magnesium and BMD due to an increase in pro-inflammatory factors (Toba et al., 2000). In the experimental animal study, OVX rats fed with magnesium supplements had shown an increase in osteocalcin, which is a protein matrix that is considered as a marker for bone building. Our findings demonstrated higher Mg²⁺ bone concentration in the OVX + Exercise group than in the OVX group. Previous experimental animal studies have shown that there is a strong relationship between aerobic exercise such as treadmill and swimming and an increase in BMD by stimulating bone formation processes (Kang et al., 2017; Oh et al., 2016), which includes magnesium concentrations in the bones.

In our main finding, there was a notable increase in the bone magnesium level and serum magnesium level in comparison between the combination groups (OVX + RFM + Exercise) versus the control group, which supports the effectiveness of intervention in homeostasis magnesium concentration.

Our experimental results showed that ovariectomy in rats leads to a decrease the in bone potassium concentration level compared with that in the control group, potentially contributing to the development of osteoporosis. This finding is consistent with previous experimental animal studies conducted by Lei et al. (2009) and Ha et al. (2020), which reported a significant decrease in bone potassium concentrations levels in ovariectomized rats. Moreover, the present study found that OVX + Exercise group and OVX + RFM group interventions have differential effects on bone potassium concentrations in ovariectomized rats compared with the OVX group without intervention. In addition, the combination group (OVX + RFM + Exercise) had a higher bone potassium concentration level than the control group, which indicated an increase in BMD and a protective approach against osteoporosis (Ji et al., 2007).

Our findings also show a significant increase in serum potassium concentrations in the ovariectomized groups compared to that in the control group, consistent with a previous experimental study conduct by Kaastad et al. (2001), which claimed that the decrease in potassium concentrations levels in OVX rats is due to the deficiency of estrogen hormone, which causes the deficiency of the aldosterone hormone level.

Furthermore, an experimental animal study found that ovariectomy led to changes in metabolic function, specifically an increase in blood glucose levels and a decrease in insulin sensitivity. These findings suggest that the observed increase in potassium levels in the current study may be due to changes in metabolic function caused by ovariectomy (Shaban et al., 2017). Another possible explanation by Ciarambino et al. (2022) is that the increase in potassium levels in the ovariectomy group may be related to changes in renal function. Potassium levels are primarily regulated by the kidneys, and ovariectomy has been shown to alter renal function in rats, specifically a decrease in renal blood flow and the glomerular filtration rate.

In our findings, there was no significant increase or decrease in serum potassium levels in the combination group (OVX + RFM + Exercise) compared with that in the control group, suggesting that the combination of exercises and fasting may have improved effect in potassium homeostasis.

Vitamin D affects bone density, especially in the femur bones, and this effect increases when estrogen and vitamin D deficiency occur in cooperation, which leads to the high risk of fractures (Lips and van Schoor, 2011; Ali et al., 2014). This is the reason how vitamin D supplementation can prevent osteoporosis in postmenopausal women (Elwakeel et al., 2020). Vitamin D deficiency can cause imbalance in BMD levels, resulting in potassium and calcium deficiency (Lips and van Schoor, 2011). Furthermore, vitamin D suppresses the parathyroid hormone, which leads to increased calcium absorption in the intestine and deposition in the bones (Lips and van Schoor, 2011; Elwakeel et al., 2020). Our results reflect a decrease in serum levels of vitamin D in OVX rats. The relationship between OVX rats and depletion of the vitamin D level is explainable by the study of Sundell and Björnsson, who found a significant increase in the level of vitamin D in the serum of OVX rats after treatment with estrogen supplements (Sundell and Bjornsson, 1990). In another experimental animal study, there was a positive correlation between estrogen and vitamin in rats, as vitamin D increases estrogen and vice versa (Nashold et al., 2009). This process is due to the enhancement of the estrogen hormone of the vitamin D receptors in the central nervous system, which increases the absorption of vitamin D in the blood (Dzik et al., 2022).

Our study suggested that exercise intervention in the OVX + Exercise group can increase the level of vitamin D serum when compared with that in the OVX group. In agreement with our results, a randomized control trail found a significant increase in the vitamin D level in the participant after aerobic exercise (Dzik et al., 2022). In another experimental animal study conducted by Aly et al. (2016), they found a positive effect of swimming exercise on vitamin D serum among albino rats. This action may be due to the ability of exercise to increase the anabolic reaction of vitamin D in various tissues, especially in the muscle (Aly et al., 2016).

A recent study by Karras et al. (2023) investigated the effectiveness of intermittent fasting on vitamin D concentration among overweight participants. Interestingly, free 25-hydroxy vitamin D [25(OH)D] in the fasting group showed an increase that was considerable for the full study duration. In contrast to the control group, the fasting group demonstrated increased free 25(OH)D concentrations at the seventh week. Moreover, it was observed that body fat has an inverse relationship with vitamin D (Karras et al., 2023). This finding supports the present study results, which show an increase in serum vitamin D levels among the fasting group compared with the control group.

In the present study, it was found that the combination effect of RFM and exercise interventions in OVX rats eliminate the negative effects of ovariectomy and estrogen deficiency on decreasing the serum vitamin D level when compared with that in the control group.

However, in the present study, one possible explanation for the lack of effect of ovariectomy on calcium and vitamin D levels could be related to the timing of the measurements. It is possible that changes in calcium and vitamin D levels as a result of ovariectomy take longer than 1 month to manifest (Lei et al., 2009). Additionally, it is important to consider additional measures of bone health in conjunction with calcium and vitamin D levels, such as bone mineral density or bone microarchitecture. These measures may provide a more comprehensive understanding of the effects of ovariectomy on bone health in rats.

Overall, our results support the hypothesis accordant with the previous study, and this study shows significant increase in body weight and decrease in bone markers among ovariectomized rats without intervention.

In conclusion, this study demonstrated that a combination of exercise and Ramadan fasting intervention can regulate calcium, magnesium, and potassium homeostasis in femoral bone and serum concentration, in addition to maintaining bone metabolism, oxidative stress, and inflammatory status levels in the liver, kidney, and bone, which have a positive effect on bone health. The combination of the Ramadan fasting model and moderate intensity exercises could be recommended as a lifestyle modification that is protective against osteoporosis, especially in the context of depleted estrogen hormone after menopause. However, further investigation is needed to confirm the results of this study.