The Effects of Time-Restricted Eating on Inflammation and Oxidative Stress in Overweight Older Adults

Armin Ezzati; Javier A Tamargo; Leah Golberg; Mark D Haub; Stephen D Anton

doi:10.20944/preprints202412.1405.v1

Submitted:

16 December 2024

Posted:

18 December 2024

You are already at the latest version

Abstract

Time-restricted eating (TRE) has been associated with beneficial effects on inflammation and oxidative stress; however, the effects of TRE on inflammation and oxidative stress in the aging population have been unexplored. This secondary analysis tested the effects of TRE on pro-inflammatory (hs-CRP [high-sensitivity C-reactive protein], IL-1β [interleukin 1 beta], IL-6 [interleukin 6], TNF-α [tumor necrosis factor alpha]) and oxidative stress (8-isoprostane) biomarkers in ten overweight older adults (mean age = 77.1 ± 6.1 years; 6 women and 4 men), who followed a TRE protocol of 16 hours of fasting per day and consumed food ad libitum during an 8-hour window for 4 weeks. TNF-α levels decreased from 43.2 (11.2) pg/ml to 39.7 (10.0) pg/ml with a Cohen's d effect size of 0.33, and IL-1β levels decreased from 1.4 (0.8) pg/ml to 1.3 (0.6) pg/ml with a Cohen's d effect size of 0.23, suggesting potential anti-inflammatory benefits. IL-6 and hs-CRP levels showed no substantial changes (Cohen’s d ≤ 0.03). The oxidative stress marker 8-isoprostane levels decreased slightly with a Cohen's d effect size of 0.07. The findings of this pilot study provide initial insights into the potential effects of TRE on inflammatory and oxidative stress markers in older adults. Well-powered studies of longer duration are warranted to elucidate the potential benefits of TRE in aging populations.

Keywords:

inflammation

;

intermittent fasting

;

older adults

;

oxidative stress

;

time-restricted eating

Subject:

Medicine and Pharmacology - Dietetics and Nutrition

1. Introduction

Aging is accompanied by gradual biological and cellular changes that result in functional decline and significant increases in the risk of age-related diseases [1]. Among these changes is chronic inflammation, which is considered a hallmark of aging [2]. Similarly, oxidative stress, a key driver of age-related diseases, increases with aging [3]. Calorie restriction (CR) has been shown to lower inflammation and oxidative stress [4,5]. However, challenges in adherence and daily caloric intake monitoring necessitate alternative approaches [6].

Intermittent fasting (IF) is an umbrella term that describes an eating pattern in which an individual fasts for an extended time-period, typically longer than 12 hours, on a repeated basis [7]. Of the various types of IF approaches, time-restricted eating (TRE), the pattern of extending the daily fasting period and thereby reducing the daily eating period, has gained popularity over the last few years [8]. TRE has shown effectiveness as a viable dietary approach for improving body composition [9,10], metabolic parameters [10,11], inflammatory markers [12,13], and oxidative stress markers [14,15], all of which are known risk factors for several age-related chronic diseases, including diabetes, cardiovascular disease (CVD), Alzheimer's disease, and certain cancers [16,17]. Some studies reported a reduction of 200-500kcal daily with TRE without the need for calorie counting [18,19,20]. While the effects of TRE on markers of inflammation and oxidative stress in young to middle-age populations have been explored by previous studies [13,14,15], no study, to date, has specifically examined the effects of TRE, with ad libitum intake, on inflammatory markers and oxidative stress in older adults.

Therefore, the primary aim of this pilot study was to evaluate the effects of a short-term TRE intervention on changes in markers of inflammation, including tumor necrosis factor alpha (TNF- α), interleukin 1 beta (IL-1beta), interleukin 6 (IL-6), high-sensitivity C-reactive protein (hs-CRP), and markers of oxidative stress, 8-isoprostane, in overweight, older adults. We hypothesized that short-term TRE would reduce both markers of inflammation and oxidative stress in overweight, older adults.

2. Materials and Methods

2.1. Study Design

This pilot study aimed to assess the effects of TRE on inflammatory markers (IL-1β, IL-6, TNF-α) and oxidative stress (8-isoisoprostane) in 10 overweight, older adults from a single-arm intervention, as previously described [21]. The primary goal of the study was designed to assess the feasibility and safety of TRE in overweight, sedentary older adults over a four-week period. Participants were included if they were aged ≥ 65 years, self-reported difficulty walking ¼ mile or climbing a flight of stairs, were sedentary (<30 minutes of structured exercise per week), had a walking speed <1 m/sec on the 4 m walk test, and had a body mass index between 25–40 kg/m² (inclusive).

2.2. Intervention

Study participants were asked to fast for a target of 16 hours per day for a period of 4 weeks while they were allowed to eat ad libitum during the daily 8-hour eating window. The first week involved a ramp-up to a full 16-hour fasting period (days 1-3 fast for 12-14 hours per day, days 4-6 fast for 14-16 hours per day, days 7-28 fast for 16 hours per day). Participants were allowed to consume calorie-free beverages, tea, black coffee, and sugar-free gum, and were encouraged to drink plenty of water throughout the intervention period. Participants were asked to record the time of first and final food/drink consumption each day. All testing sessions were performed at the Institute on Aging, University of Florida. The fasting times were self-selected by participants and were allowed to vary daily.

2.3. Measures

Fasting blood samples were collected at baseline and the 4-week follow-up visits for inflammatory and oxidative stress biomarkers. Inflammatory cytokines, including IL-1β, IL-6, and TNF-α, were assessed using the Magnetic Luminex Assay Human Premixed Multi-Analyte Kit (R&D Systems, Minneapolis, MN, USA). Human Quantikine ELISA (R&D Systems) was performed to measure hs-CRP levels. The oxidative stress marker 8-isoprostane was measured by 8-iso-Prostaglandin (CELL BIOLABS, San Diego, CA, USA).

2.4. Statistical Analysis

Data were checked for missing values and outliers. Any missing data, which occurred due to values falling below the detection limit, were excluded from the analysis. Means and standard deviations (SD) were calculated for each marker per time-point. Primary analyses were performed using SAS software, version 9.4 (SAS Institute Inc., Cary, NC, USA). Statistical analysis included paired sample t-tests to compare baseline and week four values for each marker. Changes from baseline to week four were calculated for each outcome and expressed as mean difference and percent change. Effect sizes were calculated using Cohen's d to measure the magnitude of the changes. Given the small sample size and focus on feasibility in this pilot study, hypothesis testing may not be adequate and can be misleading. As such, we report p-values but warrant caution in their interpretation.

3. Results

3.1. Changes in Inflammatory and Oxidative Stress Markers

Figure 1 shows the changes in inflammatory and oxidative stress markers from baseline to the end of 4-weeks of time-restricted eating. There were modest reductions in IL-1b and TNF-α, with effect sizes ranging between 0.23-0.33. We did not observe similar changes in other markers of inflammation of oxidative stress. Table 1 reports changes in inflammatory and oxidative stress markers from baseline to the end of 4 weeks. No significant changes were observed in any of the markers at the 4-week time point (all p-values > 0.05).

4. Discussion

To our knowledge, this is the first study investigating the effects of TRE with ad libitum intake on markers inflammation and oxidative stress specifically in sedentary, overweight older adults. The main finding of this study was that TRE produced modest reductions in two proinflammatory makers, namely TNF-alpha and IL-1B (based on effect sizes) but did not induce changes in other makers of inflammation, CRP and IL-6, or oxidative stress (8-isoprostane). Given the short-term nature of this study and small sample size, it is possible that TRE may significantly reduce markers of inflammation in adequately powered and longer-duration studies among older populations.

Previous studies have examined the effects of TRE on proinflammatory markers in adults with or without obesity [12,13,14,15,22]. In a short-term randomized controlled trial (RCT) [12], TNF-α significantly decreased only with early TRE (6 a.m.–3 p.m.) but not with mid-day TRE (11 a.m.–8 p.m.) in young, healthy individuals without obesity. In another short trial, Sutton et al. tested early TRE (last meal before 3:00 p.m.) in men with prediabetes over five weeks and observed no significant changes in IL-6 and hs-CRP levels [15]. Additionally, an eight-week trial of late TRE with longer fasting durations (18 and 20 hours) did not alter TNF-α or IL-6 compared to a control in adults with obesity [14].

In contrast to short-term studies, a longer-term study of one-year duration reported a significant reduction in TNF-α, IL-1β, and IL-6 with late TRE (three meals: 1 p.m., 4 p.m., and 8 p.m.) compared to a control group in healthy resistance-trained males [13]. These findings align with the modest improvements in TNF-α and IL-1β observed in our study which imposed self-selected eating windows with TRE. Specifically, our four-week TRE intervention showed small effect sizes for reductions in TNF-α and IL-1β, suggesting that even a shorter duration of TRE may initiate positive shifts in inflammatory markers, particularly in aging populations. While these effects were modest and non-significant, they highlight the potential of TRE to impact systemic inflammation over time. The small effect sizes observed may illustrate early, subtle changes in inflammatory pathways that could potentially induce more pronounced effects with longer interventions or higher adherence to TRE protocols. Furthermore, these findings highlight the possibility that older adults, who often exhibit heightened levels of chronic, low-grade inflammation (inflammaging), may respond differently to TRE compared to younger populations.

Our null findings with IL-6 and hs-CRP are also consistent with some prior short-term studies. Martens et al. [23] examined eucaloric TRE (16:8), without weight loss, in 22 healthy non-obese middle-age and older adults in a RCT. After 6 weeks, there were no significant differences between TRE and normal diet control for IL-6 and CRP levels [34]. Similarly, others have found no significant changes in IL-6 and CRP/ hs-CRP levels with short-term TRE interventions (5 to 8 weeks) [11,14,15]. In contrast, early 16:8 TRE (eating window between 8a.m-4 p.m.) resulted in significant reductions in hs-CRP levels (42%) in overweight and obese women with polycystic ovary syndrome over 5 weeks [22].

To our knowledge, the present study is the first to examine a marker of oxidative stress following 16:8 TRE. Oxidative stress marker 8-isoprostane slightly decreased after 4 weeks of TRE. In contrast, longer daily fasting durations of 18-20 hours have been shown to lower 8-isoprostane significantly in prediabetic men or adults with obesity after 5 and 8 weeks, respectively [14,15]. Notably, 18:6 early TRE significantly reduced the oxidative stress marker 8-isoprostane, even in the absence of weight loss [15]. Additionally, late TRE with longer fasting durations (18 and 20 hours) reduced oxidative stress 8-isoprostane in adults with obesity following 8 weeks [15]. In the secondary analysis of the study [24], however, reductions in oxidative stress were more closely related to the degree of weight and fat loss (>3.5% weight loss). Our study observed lower weight loss (~3% of initial body weight), implemented 16-hour TRE, and had a shorter time frame, which may partly explain the unchanged levels of oxidative stress.

4.1. Future Directions

The current pilot study utilized self-selected eating windows, with the average eating time occurring 61 minutes later at week 4 compared to week 1 (mean start time: 9:32 a.m. in week 1 vs. 10:33 a.m. in week 4) [25]. In contrast, a 5-week early TRE intervention (6 a.m. to 3 p.m.) improved pro-inflammatory cytokines (TNF-α and IL-8) in young adults without obesity, while mid-day TRE (11 a.m. to 8 p.m.) did not show improvements in these markers [12]. Future research should investigate the effects of early TRE and different meal timings within TRE protocols on the aging population.

In addition to meal timing, fasting duration may also affect changes in makers of inflammation and oxidative stress. However, the current study examined a 16-hour TRE, which may not have been sufficient to fully capture the effects within this study's shorter timeframe. Future well-powered studies with longer fasting durations could provide further clarity on the optimal timing and duration to maximize the health benefits of TRE in older adults. Additionally, the effects of TRE interventions on other hallmarks of aging including autophagy, epigenetic alterations, and cellular senescence should be investigated in future trials.

4.2. Strengths and Limitations

This was the first trial to explore the effects of TRE with ad libitum intake on inflammation and oxidative stress in older adults. The strength of this study is the reporting of effect sizes, which provide a nuanced understanding of the potential impact of TRE beyond statistical significance. The inclusion of effect size analysis is particularly valuable in pilot studies with small sample sizes helping to access the magnitude of observed changes for designing of future larger trials. The study is also strengthened by high levels of adherence, which may be partly due to self-selected eating windows. Participants reported an adherence rate of 84% through self-reported diaries [25].

The study has several limitations, and the pilot nature of the study, focus on feasibility, and short duration warrant careful interpretation of the results. Although common in dietary interventions, self-reporting can introduce potential limitations due to recall bias and social desirability bias. Additionally, while self-selected eating windows may enhance adherence to TRE, it may also hinder the ability to assess the effects of meal timing (early, mid-day, late) on health outcomes. Lastly, the lack of dietary intake assessment limited our ability to control confounding factors that could influence the effects of dietary interventions such as TRE. These limitations call for longer, more robust trials with comprehensive dietary assessments and advanced monitoring tools to better elucidate the impact of TRE in mitigating age-related inflammation and oxidative stress.

5. Conclusions

In conclusion, this pilot study provides initial insights into the potential effects of TRE on inflammation and oxidative stress in overweight older adults. While the small effect sizes observed suggest early, subtle changes in inflammatory pathways, the findings underscore the feasibility of TRE in this population, supported by high adherence rates. However, given the small sample size and short duration of the study, this finding should be interpreted with caution. Future well-powered studies with longer durations are needed to elucidate the effect of TRE on inflammation and oxidative stress in older populations.

Author Contributions

The authors’ responsibilities were as follows: A.E. and S.D.A. conceptualized and designed the research ("Conceptualization"); A.E., J.A.T., and L.G. conducted the analyses; A.E. wrote the manuscript; S.D.A., J.A.T., and M.D.H. critically reviewed the manuscript; and A.E. and S.D.A. had primary responsibility for the final content. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

This study was reviewed and approved by the Institutional Review Board of the University of Florida.

Informed Consent Statement

Written informed consent was obtained from all participants involved in the study.

Data Availability Statement

The data presented in this manuscript may be available upon request, subject to the submission of an application and approval by the corresponding author.

Acknowledgments

The authors extend their gratitude to the participants and research associates whose contributions were essential to the successful completion of this research project. This study was supported by funding from the NIH Claude D. Pepper Older Americans Independence Center (P30AG028740).

Conflicts of Interest

The authors declare no conflicts of interest.

References

Guo, J.; Huang, X.; Dou, L.; et al. Aging and aging-related diseases: from molecular mechanisms to interventions and treatments. Sig Transduct Target Ther. 2022, 7, 391. [Google Scholar] [CrossRef] [PubMed]
López-Otín, C.; Blasco, M.A.; Partridge, L.; Serrano, M.; Kroemer, G. Hallmarks of aging: An expanding universe. Cell. 2023, 186, 243–278. [Google Scholar] [CrossRef] [PubMed]
Liguori, I.; Russo, G.; Curcio, F.; et al. Oxidative stress, aging, and diseases. Clin Interv Aging. 2018, 13, 757–772. [Google Scholar] [CrossRef] [PubMed]
Kökten, T.; Hansmannel, F.; Ndiaye, N.C.; et al. Calorie Restriction as a New Treatment of Inflammatory Diseases. Adv Nutr. 2021, 12, 1558–1570. [Google Scholar] [CrossRef]
Bosch-Sierra, N.; Grau-del Valle, C.; Salom, C.; et al. Effect of a Very Low-Calorie Diet on Oxidative Stress, Inflammatory and Metabolomic Profile in Metabolically Healthy and Unhealthy Obese Subjects. Antioxidants. 2024, 13, 302. [Google Scholar] [CrossRef]
Dorling, J.L.; Das, S.K.; Racette, S.B.; et al. Changes in body weight, adherence, and appetite during 2 years of calorie restriction: the CALERIE 2 randomized clinical trial. Eur J Clin Nutr. 2020, 74, 1210–1220. [Google Scholar] [CrossRef]
Ezzati, A.; Rosenkranz, S.K.; Phelan, J.; et al. The Effects of Isocaloric Intermittent Fasting vs Daily Caloric Restriction on Weight Loss and Metabolic Risk Factors for Noncommunicable Chronic Diseases: A Systematic Review of Randomized Controlled or Comparative Trials. J Acad Nutr Diet. 2023, 123, 318–329.e1. [Google Scholar] [CrossRef]
Ezzati, A.; McLaren, C.; Bohlman, C.; et al. Does time-restricted eating add benefits to calorie restriction? A systematic review. Obesity (Silver Spring). 2024, 32, 640–654. [Google Scholar] [CrossRef]
Sampieri, A. , Paoli, A., Spinello, G. et al. Impact of daily fasting duration on body composition and cardiometabolic risk factors during a time-restricted eating protocol: a randomized controlled trial. J Transl Med 2024, 22, 1086. [CrossRef]
Lin S, Cienfuegos S, Ezpeleta M.; et al. Time-restricted eating without calorie counting for weight loss in a racially diverse population: a randomized controlled trial. Ann Intern Med. 2023, 176, 885–895.
Wilkinson, M.J.; Manoogian, E.N.C.; Zadourian, A.; et al. Ten-Hour Time-Restricted Eating Reduces Weight, Blood Pressure, and Atherogenic Lipids in Patients with Metabolic Syndrome. Cell Metab. 2020, 31, 92–104.e5. [Google Scholar] [CrossRef] [PubMed]
Xie, Z.; Sun, Y.; Ye, Y.; et al. Randomized controlled trial for time-restricted eating in healthy volunteers without obesity. Nat Commun. 2022, 13, 1003. [Google Scholar] [CrossRef] [PubMed]
Moro, T.; Tinsley, G.; Pacelli, F.Q.; Marcolin, G.; Bianco, A.; Paoli, A. Twelve Months of Time-restricted Eating and Resistance Training Improves Inflammatory Markers and Cardiometabolic Risk Factors. Med Sci Sports Exerc. 2021, 53, 2577–2585. [Google Scholar] [CrossRef] [PubMed]
Cienfuegos S, Gabel K, Kalam F; et al. Effects of 4- and 6-h time-restricted feeding on weight and cardiometabolic health: a randomized controlled trial in adults with obesity. Cell Metab. 2020, 32, 366–378.e3. [CrossRef]
Sutton, E.F.; Beyl, R.; Early, K.S.; et al. Early time-restricted feeding improves insulin sensitivity, blood pressure, and oxidative stress even without weight loss in men with prediabetes. Cell Metab. 2018, 27, 1212–1221.e3. [Google Scholar] [CrossRef]
Ezzati, A.; Pak, V.M. The effects of time-restricted eating on sleep, cognitive decline, and Alzheimer's disease. Exp Gerontol. 2023, 171, 112033. [Google Scholar] [CrossRef]
Ezzati, A.; Rosenkranz, S.K.; Horne, B.D. Importance of Intermittent Fasting Regimens and Selection of Adequate Therapy on Inflammation and Oxidative Stress in SARS-CoV-2 Infection. Nutrients. 2022, 14, 4299. [Google Scholar] [CrossRef]
Gabel, K.; Hoddy, K.K.; Haggerty, N.; et al. Effects of 8-hour time restricted feeding on body weight and metabolic disease risk factors in obese adults: a pilot study. Nutr Healthy Aging. 2018, 4, 345–353. [Google Scholar] [CrossRef]
Haganes, K.L.; Silva, C.P.; Eyjólfsdóttir, S.K.; et al. Time-restricted eating and exercise training improve HbA1c and body composition in women with overweight/obesity: a randomized controlled trial. Cell Metab. 2022, 34, 1457–1471.e4. [Google Scholar] [CrossRef]
Peters, B.; Vahlhaus, J.; Pivovarova-Ramich, O. Meal timing and its role in obesity and associated diseases. Front Endocrinol. 2024, 15, 1359772. [Google Scholar] [CrossRef]
Anton, S.D.; Lee, S.A.; Donahoo, W.T.; McLaren, C.; Manini, T.; Leeuwenburgh, C.; Pahor, M. The Effects of Time Restricted Feeding on Overweight, Older Adults: A Pilot Study. Nutrients. 2019, 11, 1500. [Google Scholar] [CrossRef] [PubMed]
Li, C.; Xing, C.; Zhang, J.; Zhao, H.; Shi, W.; He, B. Eight-hour time-restricted feeding improves endocrine and metabolic profiles in women with anovulatory polycystic ovary syndrome. J Transl Med. 2021, 19, 148. [Google Scholar] [CrossRef] [PubMed]
Martens, C.R.; Rossman, M.J.; Mazzo, M.R.; Jankowski, L.R.; Nagy, E.E.; Denman, B.A.; Richey, J.J.; Johnson, S.A.; Ziemba, B.P.; Wang, Y.; Peterson, C.M.; Chonchol, M.; Seals, D.R. Short-term time-restricted feeding is safe and feasible in non- obese healthy midlife and older adults. GeroScience. 2020, 42, 667–686. [Google Scholar] [CrossRef] [PubMed]
Akasheh, R.T.; Ankireddy, A.; Gabel, K.; Ezpeleta, M.; Lin, S.; Tamatam, C.M.; Reddy, S.P.; Spring, B.; Cheng, T.-Y.D.; Fontana, L.; et al. Effect of Time-Restricted Eating on Circulating Levels of IGF1 and Its Binding Proteins in Obesity: An Exploratory Analysis of a Randomized Controlled Trial. Nutrients 2024, 16, 3476. [Google Scholar] [CrossRef]
Lee, S.A.; Sypniewski, C.; Bensadon, B.A.; McLaren, C.; Donahoo, W.T.; Sibille, K.T.; Anton, S. Determinants of adherence in time-restricted feeding in older adults: lessons from a pilot study. Nutrients. 2020, 12, 874. [Google Scholar] [CrossRef]

Figure 1. Box-and-whisker plots showing the distribution of data for inflammatory markers and oxidative stress marker at baseline and after 4 weeks of time-restricted eating (TRE). The plots are labeled as follows: (a) TNF-α, Tumor Necrosis Factor alpha), (b) IL-1β (Interleukin-1 beta), (c) IL-6 (Interleukin-6), (d) hs-CRP (high-sensitivity C-reactive protein), and (e) 8-Isoprostane.

Table 1. Changes in inflammatory and oxidative stress markers from baseline to 4 weeks of time-restricted eating (TRE).

Study Measures	Baseline M (SD)	TRE M (SD)	Δ Mean (SD)	%Change	Cohen’s d	p-Value
TNF-alpha (pg/ml)	43.2 (11.2)	39.7 (10.0)	−3.5 (5.7)	−8%	0.33	0.101
IL-1β (pg/ml)	1.4 (0.8)	1.3 (0.6)	−0.1 (0.7)	−11%	0.23	0.499
IL-6 (pg/ml)	10.8 (7.2)	10.8 (7.2)	−0.001 (2.4)	0%	0.03	0.998
hs-CRP (mg/L)	3.3 (4.1)	3.6 (3.4)	0.3 (1.8)	10%	0.01	0.596
8-isoprostane (ng/mL)	41.3 (13.8)	38.7 (11.6)	-0.4 (12.2)	-1%	0.07	0.919

Table 1: Changes in inflammatory and oxidative stress markers from baseline to the end of 4 weeks of time-restricted eating (TRE). Values are presented as mean (standard deviation), with Δ Mean representing the difference from baseline. Percent change reflects the relative change from baseline to TRE. Cohen’s d (effect size) indicates the magnitude of change, and p-value denotes statistical significance.

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