Abstract

Background:

Prophylactic probiotics and intermittent fasting (IF) substantially modulate the neuropsychological functions and exercise capacity in rats subjected to sleep deprivation (SD). A comparative study was conducted to analyze the effects of probiotics and IF on SD-induced neuropsychological disturbances and compromised muscle endurance.

Methods:

Forty albino Wistar rats were randomly assigned to four groups. The NSD group was maintained on a standard chow diet for 12 weeks. The SD group followed an SD regimen for 72 h per week over 8 weeks, starting from the fifth week. The SDP group received probiotics at a dose of colony-forming units (CFUs)/100 g/day for 4 weeks prior to SD, followed by 8 weeks of concurrent probiotic administration with SD. The SDIF group underwent an alternate-day fasting regimen for 4 weeks before SD, followed by 8 weeks of simultaneous SD combined with IF. Neuropsychological functions and exercise capacity were tested, and then the brains were carefully dissected, sectioned, and processed for hematoxylin and eosin, cresyl violet, and immunohistochemical staining.

Results:

Inflammatory markers, including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and hippocampal expression of the circadian locomotor output cycles kaput (CLOCK) gene, were significantly elevated in the SD group. Conversely, it showed significant decreases in endurance, exploratory behavior, hippocampal superoxide dismutase (SOD) activity, and fecal short-chain fatty acids (SCFAs). Histological analysis also revealed hippocampal gliosis, apoptosis, CA1 pyramidal cell degeneration, layer disorganization, and upregulation of glial fibrillary acidic protein (GFAP), NF-κB, and cleaved caspase-3. Nevertheless, both probiotics and IF markedly reduced serum MDA, hippocampal CLOCK gene expression, gliosis, and apoptosis and enhanced memory performance. In addition, they significantly increased hippocampal SOD activity and SCFAs.

Conclusion:

These findings indicate that prophylactic probiotics decrease cognitive disruption and impaired muscle endurance caused by SD through CLOCK gene regulation compared to that with IF. This highlights the need for further research to elucidate these mechanisms. Histological findings also supported these results, showing improved neuronal structure in the hippocampus following probiotic treatment.

Rats were randomly placed in the NSD, SD, SDP, and SDIF groups. During the experiment, this study evaluated the exercise performance and behavior analyses; measured fecal SCFAs, serum analyses, and hippocampal gene expression; and conducted histopathology and immunohistochemical analyses. NSD, non-sleep-deprivation group; SD, sleep deprivation; SDP, sleep-deprived on probiotics regimen group; SDIF, sleep-deprived on intermittent fasting regimen group.

1 Introduction

Sleep deprivation (SD) has been linked to a poor quality of life (Chung et al., 2023), and there is an approximately 14% higher risk of mortality for individuals sleeping less than 7 h per night (Ungvari et al., 2025). SD is a severe stressor, and its allostatic load can lead to cognitive impairments (McEwen, 2006) and reduced muscular endurance (Rault et al., 2024). Chronic SD is connected to cardiovascular complications (Evbayekha et al., 2022), cognitive decline (Han et al., 2024), metabolic imbalances (Jain et al., 2012), and a compromised immune response (Garbarino et al., 2021). These impairments lead to unhealthy aging and age-related pathologies such as diabetes, dementia, and cancer (Carroll and Prather, 2021). It enhances oxidative stress, increases inflammation, and causes hormonal imbalances (Ungvari et al., 2025). In addition to disrupting natural circadian cycles, SD alters the expression of core clock genes, which are crucial for maintaining immunological homeostasis (Zeng et al., 2024). SD disrupts the rhythmic expression of the central and peripheral clock genes, including CLOCK and BMAL1, causing dysregulation of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) (Rodrigues et al., 2023; Huang et al., 2025).

Probiotics are “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host (Sanders, 2008). These are types of bacteria, such as Lactobacillus, Bifidobacterium, and Saccharomyces, that are found in dietary supplements, fermented foods, or specialized products. It may improve the body’s health by modulating gut microbiota, regulating epithelial gut barrier integrity, and enhancing immune responses (Amin et al., 2025). It may also improve lactose digestion and manage inflammatory bowel disease, metabolic syndrome, and even neuropsychiatric conditions via the gut–brain axis (GBA). Given the established potential of gut microbiota modulation to influence neurological outcomes, an established blend of Lactobacillus strains (L. fermentum and L. delbrueckii) at a dosage validated by Liu et al. (2019) was implemented 4 weeks before the SD to ensure sufficient microbiota modulation, allowing us to evaluate the preventative potential of these probiotics against SD-induced deficits via the GBA.

Probiotics’ mechanisms of action could modulate cytokine profiles and short-chain fatty acid (SCFA) production (Plaza-Diaz et al., 2019). Probiotics are also recognized for their systemic and neurocognitive effects (Lin F. L. et al., 2023). Probiotics can alleviate sleep disturbance-related mood symptoms (Liu et al., 2025) by enhancing SCFA production, modulating neurotransmitter pathways (serotonin and GABA), and strengthening the intestinal and blood–brain barriers (Li et al., 2024). Probiotics have been administered to improve sleep quality (Irwin et al., 2020).

On the other hand, intermittent fasting (IF), a diet plan that includes alternating periods of eating and abstaining from food, involves popular regimens such as alternate-day fasting, the 5:2 plan, and time-restricted feeding (TRF) (Patterson and Sears, 2017). IF supports the loss of weight, improves insulin sensitivity, enhances metabolic health, and reduces the risk of heart disease (Vasim et al., 2022). Furthermore, IF affects circadian rhythms (Daas and de Roos, 2021), oxidative stress, and inflammatory processes (Reddy et al., 2024). Moreover, IF affects the gut microbiome (Liu et al., 2021), helping beneficial bacteria grow and increasing the production of SCFAs, which enhance the immunological and neurocognitive functions (Hein et al., 2025). These findings indicate that IF improves nutrition and is a potential treatment that can affect the body.

Thus, in the current study, we aimed to compare the prophylactic effects of probiotics and IF on neuropsychological functions and exercise capacity, their relationship to the CLOCK gene in experimentally induced SD in rats, and the possible molecular mechanisms mediating their action by using various physiological, histological and immunohistochemical methods.

2 Materials and methods

The experimental protocol obtained endorsement under approval number 6/2023PHYS16 from the local ethics committee at the Faculty of Medicine, Menoufia University. This was conducted based on the guidelines delineated in the Guide for the Care and Use of Laboratory Animals (National Academies Press, eighth edition) (Council et al., 2010).

3.1 Experimental design

Forty adult male Wistar albino rats weighing between 100 and 150 g and aged between 5 and 6 weeks were used in this study. Throughout the study, all rats had unrestricted access to food and water after a 2-week conditioning period under stable environmental conditions, temperature of 22 °C ± 2 °C, and humidity of 40%–70% with a 12:12-h light/dark cycle (lights on at 07:00). The rats were divided into four experimental groups (10/group):

Non-sleep deprivation group (NSD): In this group, rats were administered a conventional rat chow diet over a duration of 12 weeks (Hazzaa et al., 2020).

Sleep deprivation group (SD): In this group, rats were sleep deprived for a total of 72 h SD per week for 8 weeks, starting from the fifth week, by the modified multiple platform method (MMPM) (Chung et al., 2023).

Sleep-deprived on probiotics regimen group (SDP): In this group, before being subjected to SD, rats were administered probiotic supplements for four consecutive weeks at a dosage of one billion colony-forming units (CFUs)/100 g/day of Lactobacillus fermentum and Lactobacillus delbrueckii by oral gavage; this choice and regimen are strategically based on the robust protocols established in prior literature (Liu et al., 2019; Azagra-Boronat et al., 2020) to ensure the symbiotic benefits of these species. Then, 8-week chronic SD (total of 72 h SD per week), as in the SD group, was induced continually and simultaneously with probiotics for the subsequent weeks.

Sleep-deprived on intermittent fasting regimen group (SDIF): In this group, before being subjected to SD, rats followed a dietary regimen IF) in which food was available only every other day for four consecutive weeks (Hazzaa et al., 2020). Then, 8-week chronic SD (total 72 h SD per week), as in the SD group, was induced continually and simultaneously with IF for the subsequent weeks (Figure 1).

Experimental timeline illustrating a 4-week pretreatment phase followed by 8 weeks of chronic sleep deprivation. Rats were assigned to four groups, namely, normal sleep (NSD), sleep deprivation (SD), sleep deprivation with probiotic treatment (SDP), and sleep deprivation with intermittent fasting (SDIF).

3.2 Sampling

Behavioral assessments (using open-field test, Y-maze, and tail suspension test) and endurance evaluations were conducted, with fecal specimens being acquired for SCFA analysis at the initiation of the study, the conclusion of the fourth and eighth weeks, and after 12 weeks (marking the experiment’s conclusion) from the onset of the study. The subjects underwent an overnight fasting period, subsequent to which the samples of blood were procured from the retro-orbital venous plexus. Serum samples were subsequently isolated and utilized for the quantification of serum pro-inflammatory markers, specifically IL-6, (TNF-α), and malondialdehyde (MDA). The rats were euthanized via cervical decapitation under anesthesia (achieved through an intraperitoneal administration of 60 mg/kg phenobarbital), and their brains were classified into two segments. One segment was placed at −80 °C for the relative quantification of circadian locomotor output cycles kaput CLOCK gene expression within hippocampal tissues and for the evaluation of superoxide dismutase (SOD) activity. The other segment was placed in 10% neutral formalin for conducting histopathological and immunohistochemical analyses.

3.3 Assessment of behavioral responses

Evaluation was conducted between the hours of 9:00 a.m. and 2:00 p.m. within a quiet room with natural daylight. Before conducting evaluations, the rats were submitted to the observation environment for 1 hour. Each test was controlled using a video recording device, which was a Samsung ST93 Digital Camera manufactured in Suwon, South Korea. The apparatus was sanitized using 70% ethanol to alleviate any olfactory cues that may affect the subjects before each evaluation (Matheus et al., 2016).

3.4 Assessment of motor function3.4.1 Swimming endurance test

The rats were put in a swimming apparatus (100 cm in diameter × 40 cm in depth) full of water maintained at a temperature of 25 °C ± 2 °C to a depth of 30 cm, with weights (5% of body mass) affixed to the tail base of each rat (Xu et al., 2018). The duration of exhaustive swimming was recorded based on the observation of uncoordinated movements in an individual rat, wherein the rat failed to resurface within a timeframe of 5 s.

3.4.2 Open-field test

A wooden arena (100 cm × 100 cm × 60 cm in height, featuring light brown walls and flooring) was classified into 25 squares. The rat was placed in the open arena and permitted to navigate freely for a span of 10 min (Wang et al., 2023a). The number of squares traversed, frequency of grooming behaviors, instances of rearing, total entries into the central zone, and occurrences of freezing behavior were quantified, along with the duration spent in the inner and outer zones.

3.5 Assessment of short-Term spatial working memory3.5.1 Y-maze

The Y-maze consists of three arms, extending approximately 50 cm in length, 10 cm in width, and 20 cm in height. This apparatus evaluates spatial working memory capabilities (Soares et al., 2013). A single rodent was placed near the maze’s center and permitted to explore the Y-maze for a period of 8 min. To facilitate spatial orientation, various distal visual cues (e.g., geometric shapes) were positioned on the room walls around the Y-maze and retained in fixed locations throughout the testing period. Spatial cognition was assessed by calculating the percentage of spontaneous alternation, which is calculated as (the number of successful alternations/(the total number of entries – 2) x 100.

3.6 Assessment of depressive-like behavior3.6.1 Tail suspension test

The tail suspension test (TST) is a predictive measure for depressive-like behavior. Rats were hung 1 cm from the tip by their tails for a short duration (6 min) at a height of 25 cm above the ground (Can et al., 2012; Pignataro et al., 2023). The period of immobility was recorded, which is defined as the state where the rat exhibited passivity without any observable movement. Adult male Wistar albino rats weighing between 100 and 150 g and aged between 5–6 weeks were used in this study. This specification was chosen to ensure the safety and suitability of the test.

3.6.2 Biochemical analysis

The serum IL-6 value was measured using ELISA commercial kits (DI develop, Canada) according to Brianza-Padilla et al. (2018).

The serum TNF-α was estimated using ELISA kits (Biotech, Shanghai, China) according to Li et al. (2020).

The serum MDA concentration was quantified using spectrophotometric methods combined with commercially available kits (Biodiagnostic Company, Cairo, Egypt) according to the protocol delineated by Draper et al. (1993).

The fecal SCFA was measured by gas chromatography–mass spectrometry (GC–MS) after derivatization to the corresponding pentafluorobenzyl bromide (PFBBr) derivative, as detailed by Hussein et al. (2014).

3.6.3 Measurement of hippocampal tissues SOD

Hippocampal tissues were homogenized in a normal saline solution at a ratio of 1:9 (w/v). The resultant homogenate was centrifuged at 1,800 g/min for 10 min. The supernatant was used to quantify SOD. The concentration of SOD was evaluated spectrophotometrically using commercial kits (Biokit Company, Egypt) according to the illustrated methodology in Liu et al. (2007).

3.7 Quantitative assay of CLOCK gene expression using the reverse transcriptase polymerase chain reaction technique (RT-PCR)

Hippocampal tissues were used for the isolation of RNA using the PureLinkTM RNA Mini Kit (Life Technologies). The quality and purity of the RNA were confirmed. The RNA was stored at −80 °C until further use. The first stage involved the synthesis of complementary DNA (cDNA) using the G-Storm Thermal Cycler (United Kingdom) combined with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, United States) for a single cycle. Primers specific to GAPDH were used as an RNA loading control during RT-PCR reactions. The second stage included the amplification of cDNA; cDNA was utilized in SYBR green-based quantitative real-time PCR for the relative quantification (RQ) of circadian locomotor output cycles kaput (CLOCK) gene expression, facilitated by the SensiFASTTM SYBR Lo-ROX Kit (United States), utilizing the following specifically designed primers (Biokit, Egypt): the forward primer for CLOCK was (5-TCA​CCA​CGT​TCA​CTC​AGG​ACA-3) and the reverse primer was (5-AAGGATTCCCA TGGAGC AA-3). The analysis was performed using Applied Biosystems 7500 software version 2.0.1. The RQ of the CLOCK gene expression was defined using the comparative ΔΔCt method, where the CLOCK gene mRNA was normalized to an endogenous reference gene (GAPDH) compared to a control, which is pursuant to the guidelines established in Koritala et al. (2021).

3.8 Histological studies

Brain tissues were removed, and parasagittal sections were prepared. The samples were fixed in a 4% solution of formaldehyde for 24 h after dehydration through a graded series of increasing alcohol concentrations. The tissues were subjected to a clearing process using xylene and were embedded in paraffin blocks. The paraffin-embedded brain blocks were sectioned into to a thickness of 4 μm, deparaffinized, and affixed to glass slides for the following procedures:

Hematoxylin and eosin (H&E) staining was performed to illustrate the history of architecture of the hippocampus, as described by Shalaby et al. (2024).

Cresyl violet staining was performed to determine viable surviving neurons that were defined by spherical open-faced nuclei, as indicated by Suvarna et al. (2018).

Immunohistochemical staining was conducted using antibodies against glial fibrillary acidic protein (GFAP) as an astrocytic marker for the assessment of gliosis; cleaved caspase-3 as an indicator of apoptosis; and nuclear factor-kappa β (NF-kβ), IL-6, and TNF-α as markers of inflammation, as described by Fareed et al. (2022) and Shalaby et al. (2023).

The slides were incubated in a 3% solution of hydrogen peroxide in phosphate-buffered saline (PBS) for 30 min to mitigate endogenous peroxidase activity. Antigen retrieval was performed by heating the slides in sodium citrate (10 mM, pH 6.0) at 95 °C for 15 min. They were incubated for 1 h at room temperature in PBS supplemented with 10% normal serum to block non-specific binding.

The slides were incubated overnight at 4 °C with the following antibodies: cleaved caspase-3 (rabbit polyclonal, 1:300, #9662, Cell Signaling Technology, Danvers, MA, United States), GFAP (mouse monoclonal, 1:200, SC-58766, Santa Cruz Biotechnology, Inc.), IL-6 (rabbit polyclonal, 1:1,200, Wako, Osaka, Japan), TNF-α (rabbit polyclonal, Catalog No. A11534, ABclonal Technology, 1:100), and NF-κB p65 (polyclonal, Catalog No. bs-20159R, Bios Antibodies).

The relevant antibodies were administered to the tissue sections after applying the streptavidin–biotin detection methodology for 20 min. The slides underwent treatment with diaminobenzidine (DAB) and were counterstained utilizing Mayer’s hematoxylin. Each section was scrutinized under a light microscope, and images were systematically captured.

The GFAP-positive cells were identified by brown staining of the cell membrane and cytoplasm responding to astrocytes.

NF-κB-positive cells, primarily pyramidal neurons, were recognized by brown staining of both the cytoplasm and nucleus of hippocampal pyramidal neurons.

TNF-α-positive cells, which are also pyramidal neurons, were identified by brown staining of the cytoplasm of hippocampal pyramidal neurons.

IL-6-positive cells were revealed by brown cytoplasmic staining in hippocampal pyramidal neurons.

Cleaved caspase-3-positive cells, which are indicative of apoptosis, were identified by brown staining of their nuclei.

3.8 Morphometric study

All slides from each animal were analyzed using a conventional light microscope equipped with a ×40 objective. For each animal, three representative slides were prepared, and five non-overlapping fields were photographed using a Nikon E400 digital microphotography system (N150, Nikon, Tokyo, Japan). A total of 10 animals per group were included in the analysis across the four experimental groups. The captured images were analyzed using Digimizer software, version 4.6.1 (MedCalc Software Ltd., Acacialaan, Belgium) (

Wang et al., 2005

). The images were analyzed to evaluate the following:

The mean number of degenerated pyramidal cells within the CA1 region of the hippocampus (H&E, ×200).

The mean number of viable pyramidal cells in the CA1 region of the hippocampus (cresyl violet, ×200).

The mean area percentage of GFAP expression (×400).

The mean area percentage of NF-κB expression (×400).

The mean area percentage of TNF-α expression (×400).

The mean area percentage of IL-6 expression (×400).

The mean number of cleaved caspase-3-positive cells (×400).

3.9 Statistical analysis

Data were shown as the mean ± SD. SPSS version 26 for Windows (SPSS Inc., Chicago, Illinois, United States) was used for statistical analysis. ANOVA and post-hoc multiple comparison tests were performed to determine the significance of group differences, and statistical significance was defined in all experimental trials when the p-value was ≤ 0.05. The SD values presented represent the variability among individual animals within the same experimental group for this single cohort. No additional independent experimental repetitions were performed; therefore, the reported variability reflects only within-cohort variation.

4 Results4.1 Swimming endurance test

The results of our study showed that SD significantly decreased the exhaustive swimming time after 8 weeks (Figure 2A, F (3, 36) = 41.150; p < 0.001) and 12 weeks (Figure 2A, F (3, 36) = 191.507; p < 0.001) from the beginning of the study relative to NSD. Moreover, probiotics significantly increased (p < 0.001) the exhaustive swimming time after 8 and 12 weeks from the beginning of the study relative to the SD and SDIF. IF insignificantly (p > 0.05) changed the exhaustive swimming time relative to SD rats throughout the experiment (Figure 2A; Table 1).

(A) Effects of probiotics and IF on exhaustive swimming time. (B) Effects of probiotics and IF on the percentage of alterations in the Y-maze (% alternation). (C). Number of crossed squares. (D). Effects of probiotics and IF on immobility time (sec) in the tail suspension test (immobility time). (E). Rearing frequency and (F). freezing episodes in OFT after 4, 8, and 12 weeks from the beginning of the study in experimentally induced SD. NSD, control group; SD, sleep deprivation; SDP, sleep deprivation with probiotics; SDIF, sleep deprivation with intermittent fasting. Error bars represent SD to indicate variability within each group.

ParameterNon-sleep-deprivation group (n = 10)Sleep deprivation group (n = 10)Sleep-deprived on probiotics regimen group (n = 10)Sleep-deprived on intermittent fasting regimen group (n = 10)Exhaustive swimming time at baseline (sec)33.40 ± 3.4133.95 ± 2.3634.06 ± 3.3434.92 ± 3.25F = 0.409, p-value = 0.748Exhaustive swimming time after 4 weeks (sec)33.49 ± 2.7433.42 ± 3.5737.44 ± 3.6933.07 ± 3.36@F = 0.3.778, p-value =0.019*Exhaustive swimming time after 8 weeks (sec)32.86 ± 3.0225.02 ± 1.83*32.58 ± 2.39$24.02 ± 1.94*@F = 41.150, p-value <0.001*Exhaustive swimming time after 12 weeks(sec)31.38 ± 1.9716.68 ± 1.46*21.71 ± 1.91*$15.02 ± 1.29*@F = 191.507, p-value <0.001*Behavioral Y-maze test% of alternations at baseline87.17 ± 4.1186.73 ± 6.1983.76 ± 6.1187.19 ± 4.03F = 0.999, p-value = 0.404% of alternations after 4 weeks84.84 ± 6.6684.16 ± 5.0091.77 ± 6.03*$88.14 ± 3.87F = 4.043, p-value =0.014*% of alternations after 8 weeks82.51 ± 5.7950.75 ± 4.93*76.61 ± 4.57$69.00 ± 7.01*$F = 59.574, p-value <0.001*% of alternation after 12 weeks86.34 ± 4.7039.48 ± 6.54*70.22 ± 6.60*$56.04 ± 4.85*$@F = 121.075, p-value <0.001*

Results of the forced swimming tests (exhaustive swimming time) and Y-maze test at different intervals among studied groups (n = 40).

Data were expressed as the mean ± standard deviation (SD); F: one-way ANOVA test, and statistical significance was taken as p ≤ 0.05 for all experiments. *: significance compared to the corresponding value of the non-sleep deprivation group, $: significance compared to the corresponding value of the sleep deprivation group, and @: significance compared to the corresponding value of the sleep-deprived on probiotics regimen group.

4.2 Y-maze test

The results of the Y-maze test showed that SD significantly decreased the percentage of alternations after 8 weeks (Figure 2B, F (3, 36) = 59.574; p < 0.001) and 12 weeks (Figure 2B, F (3, 36) = 121.075; p < 0.001) from the start of the study according to NSD rats. Moreover, probiotics increased (p < 0.05) the percentage of alternations after 4 weeks (Figure 2B, F (3, 36) = 4.043; p < 0.05), 8 weeks, and 12 weeks from the beginning of the study relative to SD rats and significantly increased (p < 0.05) the percentage of alternations after 8 and 12 weeks from the beginning of the study relative to SDIF. IF significantly (p < 0.001) increased the percentage of alterations after 8 and 12 weeks relative to the SD rats (Figure 2B; Table 1).

4.3 Open-field test

SD significantly increased rearing frequency, frequency of grooming, and freezing episodes after 8 weeks (F (3, 36) = 579.857; p < 0.001; F (3, 36) = 4.502; p < 0.05; F (3, 36) = 10.923; p < 0.001) and 12 weeks (F (3, 36) = 346.110; p < 0.001; F (3, 36) = 3.045; p < 0.05; F (3, 36) = 31.614; p < 0.001) from the start of the study compared to that in NSD rats. In contrast, it decreased the number of crossed squares and time in the center after 8 weeks (F (3, 36) = 50.538; p < 0.001; F (3, 36) = 15.401; p < 0.001) and decreased the number of crossed squares, time, and total entries to the center after 12 weeks (F (3, 36) = 77.360; p < 0.001; F (3, 36) = 82.364; p < 0.001; F (3, 36) = 17.827; p < 0.001) from the beginning of the study compared to that in NSD rats. Probiotics decreased (p < 0.05) rearing frequency, frequency of grooming, and freezing episodes after 8 and 12 weeks from the beginning of the study compared to that in the SD rats. In contrast, it significantly (p < 0.05) increased the number of crossed squares and time in the center after 4, 8, and 12 weeks from the beginning of the study compared to that in SD rats. However, IF significantly decreased (p < 0.05) rearing frequency and freezing episodes after 8 and 12 weeks from the beginning of the study relative to that in the SD rats, while it significantly (p < 0.05) increased the number of crossed squares after 4 and 8 weeks from the beginning of the study relative to that in the SD rats. Moreover, IF significantly increased (p < 0.05) rearing frequency after 8 and 12 weeks from the beginning of the study relative to the SDP rats. It significantly (p < 0.05) reduced the number of crossed squares after 4 and 8 weeks from the beginning of the study compared to that in SDP rats (Figures 2C,E,F; Table 2).

ParameterNon-sleep-deprivation group (n = 10)Sleep deprivation group(n = 10)Sleep-deprived on probiotics regimen group (n = 10)Sleep-deprived on intermittent fasting regimen group (n = 10)Number of crossed squares at baseline62.9 ± 4.8663.8 ± 5.8866.6 ± 2.8864.6 ± 5.15F = 1.070, p-value =0.374Number of crossed squares after 4 weeks61.7 ± 5.6262.8 ± 4.8079.7 ± 3.40*$70.9 ± 5.70*$@F = 28.306, p-value <0.001*Number of crossed squares after 8 weeks64.6 ± 6.4350.0 ± 3.71*59.8 ± 4.29$41.2 ± 3.52*$F = 50.538, p-value <0.001*Number of crossed squares after 12 weeks63.8 ± 5.8837.5 ± 3.06*44.8 ± 3.79*$41.2 ± 3.52*F = 77.360, p-value <0.001*Rearing frequency at baseline11.6 ± 1.2611.2 ± 1.0311.5 ± 1.7811.8 ± 1.75F = 0.281, p-value =0.839Rearing frequency after 4 weeks12.0 ± 2.2612.4 ± 2.379.80 ± 1.43$10.5 ± 1.62F = 3.923, p-value =0.016*Rearing frequency after 8 weeks13.1 ± 2.3840.0 ± 1.49*23.6 ± 1.07*$35.6 ± 1.07*$@F = 579.857, p-value <0.001*Rearing frequency after 12 weeks14.4 ± 2.7650.1 ± 3.25*29.6 ± 1.96*$44.6 ± 2.80*$#F = 346.110, p-value <0.001*Number of grooming at baseline25.6 ± 1.5127.0 ± 3.0627.0 ± 3.0628.5 ± 3.57F = 1.667, p-value =0.191Number of grooming after 4 weeks27.3 ± 2.9125.6 ± 1.6524.3 ± 2.6726.9 ± 3.31F = 2.526, p-value =0.73Number of grooming after 8 weeks29.6 ± 5.3638.4 ± 6.95*30.6 ± 5.60$33.3 ± 5.44F = 4.502, p-value =0.009*Number of grooming after 12 weeks30.1 ± 5.7438.0 ± 7.8330.4 ± 6.2434.2 ± 6.96F = 3.045, p-value =0.054Time in the center at baseline10.2 ± 1.039.80 ± 0.6310.1 ± 0.8810.0 ± 0.94F = 0.374, p-value =0.772Time in the center after 4 weeks10.1 ± 0.9910.0 ± 0.9412.0 ± 1.41*$12.0 ± 0.94*$F = 10.650, p-value <0.001*Time in the center after 8 weeks8.90 ± 1.525.80 ± 0.63*7.50 ± 1.08*$6.40 ± 0.97*F = 15.401, p-value <0.001*Time in the center after 12 weeks8.80 ± 1.402.90 ± 0.57*4.10 ± 0.88*$3.20 ± 0.79*F = 82.364, p-value <0.001*Total entries to the center at baseline6.80 ± 2.576.80 ± 2.536.70 ± 2.456.50 ± 2.42F = 0.032, p-value =0.992Total entries to the center after 4 weeks6.70 ± 2.456.70 ± 2.457.30 ± 2.166.70 ± 2.21F = 167, p-value =0.918Total entries to the center after 8 weeks4.90 ± 2.563.80 ± 1.034.50 ± 1.354.20 ± 1.32F = 0.775, p-value =0.515Total entries to the center after 12 weeks5.60 ± 2.631.20 ± 0.92*1.70 ± 1.06*81.40 ± 0.97F = 17.827, p-value <0.001*Freezing episodes at baseline15.2 ± 1.3215.4 ± 1.2615.0 ± 0.8215.1 ± 0.99F = 0.234, p-value =0.872Freezing episodes after 4 weeks15.2 ± 0.7915.2 ± 0.7912.0 ± 0.8*$213.4 ± 1.07*$@F = 31.435, p-value <0.001*Freezing episodes after 8 weeks15.1 ± 1.37