Introduction
Aging is a complex biological process characterized by a gradual decline in physiological functions and quality of life. The increasing global elderly population, associated with the rising incidence of chronic diseases, like type 2 diabetes mellitus (T2DM), necessitates effective interventions. T2DM, often linked to oxidative stress and inflammation, can lead to complications, such as non-alcoholic fatty liver disease (NAFLD), affecting over 80% of T2DM patients. Early identification of NAFLD risk through non-invasive indices, like the Fatty Liver Index (FLI), Lipid Accumulation Product (LAP), Hepatic Steatosis Index (HSI), and Framingham Steatosis Index (FSI) is crucial [9]. Exercise is a known modulator of glucose homeostasis and insulin sensitivity, with exercise timing potentially influencing metabolic responses due to circadian rhythms [10]. This study aimed to investigate whether a structured aerobic exercise program significantly improves NAFLD-related biochemical indices (FLI, LAP, HSI, and FSI) in elderly women with T2DM, and to determine if specific exercise timing (morning vs. evening) yields superior outcomes. 

Materials and Methods
This experimental study used a pre-test/post-test non-equivalent control group design in a laboratory setting. The study population included 45 women, aged 60-65 years, with T2DM from Chaharmahal and Bakhtiari province, Iran. Participants were recruited based on strict inclusion criteria, including a physician’s T2DM diagnosis, HbA1c ≥6.5%, and no prior history of NAFLD. Exclusion criteria screened for conditions that would prevent exercise or affect data validity, such as severe heart failure or neurological disorders. 
We determined a sample size of 15 participants per group (totaling 45) considering α=0.05 and 80% power, using the FLI as the primary outcome. Participants were randomly assigned to one of two exercise groups (morning or evening) or a control group. the subjects provided written informed consent. Anthropometric data and blood samples were collected before and 48 hours after the 12-week intervention. Three participants dropped out for various reasons.
Exercise groups trained for 12 weeks, with three sessions per week. Morning sessions were held at 9 AM (ZT3) and evening sessions at 9 PM (ZT15), consistent with circadian rhythm research [12]. Each 50-minute session included a warm-up, 40 minutes of brisk walking/light jogging at moderate intensity (progressively increasing from 50% to 70% of heart rate reserve), and a cool-down [20]. Heart rate was continuously monitored. 
We collected comprehensive anthropometric measurements (e.g., BMI and waist circumference) and blood samples after a 12-hour fast. We measured fasting blood sugar (FBS), serum insulin, and calculated insulin resistance (HOMA-IR) [16]. Key liver enzymes (ALT and AST), lipid profile (TG, LDL, HDL, and TC), and HbA1c levels were also assessed. NAFLD status was evaluated via sonography, and the FLI, LAP, HSI, and FSI were calculated using established formulas. 

Results
Our 12-week aerobic exercise intervention led to significant improvements in various metabolic and anthropometric markers. Inter-group analysis showed significant differences across most studied indices (P≤0.05), excluding age, height, and waist-to-hip ratio (P≥0.05). Both morning and evening exercise groups demonstrated significant improvements compared to the control group (P<0.05). Direct comparison between the two exercise groups revealed significant differences only for TG and LDL levels (P≤0.05), with no other significant differences observed (P≥0.05). Levene’s test confirmed homogeneity of variances between groups for all indices (P≥0.05). 
Both exercise groups experienced notable positive changes in blood pressure, with significant reductions in systolic and diastolic readings (P<0.001 within-group for exercise groups vs. P≥0.013 for the control group). HOMA-IR values, and FBS, insulin,  and HbA1c levels also significantly decreased in both exercise groups (P<0.001 to P<0.018 within-group), in contrast to the control group, which showed no significant change or a worsening trend. 
Liver enzymes (AST, ALT, and GGT) significantly reduced in both exercise groups (P<0.001 to P<0.005 within-group), while they increased in the control group. Furthermore, within the exercise groups, the reductions in TG, total cholesterol, and LDL levels were statistically significant, with p-values ranging from less than 0.001 to less than 0.006. Similarly, HDL levels significantly increased within these groups (P<0.001). 
Regarding NAFLD-related biochemical indices, a significant inter-group difference was observed for FLI, HSI, LAP, and FSI (P≤0.001). Both morning and evening exercise groups showed significant reductions in FLI, HSI, and FSI compared to the control group (P≤0.001). Interestingly, while both exercise groups showed a significant reduction in LAP compared to the control group, the evening exercise group exhibited a greater reduction in LAP than the morning group (P=0.030). No other significant timing-specific differences were found for FLI, HSI, or FSI. Intra-group analyses confirmed significant reductions in these indices within both exercise groups (P≤0.001), with no significant changes in the control group. 

Anthropometric outcomes
In terms of anthropometric variables, both exercise groups, unlike the control group, experienced significant decreases in weight, BMI, and body fat percentage after the intervention (P<0.05 within-group). Similarly, waist and hip circumferences significantly reduced in the exercise groups (P≤0.002 within-group). 

Conclusion 
This study aimed to evaluate the impact of aerobic exercise timing (morning vs. evening) on biochemical markers associated with NAFLD in elderly women with T2DM. Our findings demonstrated that both morning and evening aerobic exercise significantly reduced NAFLD indicators, such as FLI, HSI, LAP, and FSI compared to the control group. This highlights the effectiveness of aerobic exercise as a non-pharmacological intervention in improving liver function in T2DM patients. While most indices improved similarly across exercise groups, LAP showed a significant difference, with evening exercise yielding superior outcomes. These results align with previous research indicating the positive effects of physical activity on NAFLD [9]. The observed reductions in NAFLD indices can be attributed to multiple physiological mechanisms, including decreased visceral adipose tissue, improved insulin sensitivity, reduced TG levels, and enhanced lipid profiles. Regular exercise, particularly moderate-to-high intensity aerobic training, promotes hepatic fat oxidation and reduces inflammatory markers, thus mitigating NAFLD progression.

Ethical Considerations
Compliance with ethical guidelines
This study received approval from the Ethics Committee of the School of Health, Shahid Sadoughi University of Medical Sciences, Yazd (Cod: IR.SSU.SPH.REC.1403.093), and was registered in the Iranian Registry of Clinical Trials (Cod: IRCT20181212041935N1). 

Funding
This article was extracted from the M.Sc. thesis of Zahra Heydari Kharaji, studied at the Department of Elderly Health, Faculty of Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors.

Authors' contributions
Conceptualization, methodology, research and review, original draft writing: Zahra Heydari Kharaji; Supervision, validation, methodologist, editing and review, project management: Fatemeh Safari; Research and review, data analysis, methodology, draft writing, finalization of writing: Zahra Hemmati Farsani; Research and review, data collection, visualization: Shahrzad Habibi Ghahfarrokh; Statistical analysis, validation, editing and review: Hossein Fallahzadeh.

Conflicts of interest
The authors declared no conflicts of interest regarding the publication of this paper.

Acknowledgments
We sincerely thank all participants for their invaluable contribution and commitment to this study. Our gratitude also extends to colleagues and professors whose expertise and insights were instrumental in successfully completing this project. Finally, we appreciate the Iranian Journal of Aging’s editorial team for their meticulous review and constructive feedback, which significantly enhanced the quality of this manuscript.

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