Impact of Time-Restricted Eating and Supervised Exercise on Hepatic Steatosis and Cardiometabolic Health in Adults With Obesity
Phase: NA
Conditions: Time Restricted Feeding, Exercise, Hepatic Steatosis, Cardiometabolic Syndrome, Obesity
Sponsor: Universidad de Granada

TEMPUS, Study Protocol 
Date: 01/07/2023 
Version: 1 
 
 
 
 
TEMPUS 
 
 
 
 
 
Study Protocol 
 
 
 
 
 
University of Granada, Spain 
Trial registration number: NCT05897073 
Version: 1 
Date: 01-07-2023

TEMPUS, Study Protocol 
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Roles and responsibility: 
Name and address of principle 
Prof. Dr. Jonatan R. Ruiz (principal investigator), 
investigator and trial coordinators: 
Dr. Alba Camacho-Cardenosa (trial coordinator) 
Mr. Antonio Clavero-Jimeno (trial coordinator) 
PROFITH (PROmoting FITness and Health Through Physical 
Activity) research group, Department of Physical Education and 
Sports, Sport and Health University Research Institute (iMUDS), 
University of Granada 
Faculty of Sport Science, Carretera de Alfacar s/n, Granada, 
18071, Spain 
 
Study Team 
 
 
 
Dr. Adrián Cortés-Martín (biochemist) 
 
 
 
 
 
Dr. Alejandro De-la-O (data manager) 
 
 
 
 
 
Dr. José Luis Martín Rodríguez (radiologist) 
Dr. Manuel Muñoz-Torres (endocrinologist) 
 
 
 
 
 
Ms. Balma Boira-Nacher (nutritionist) 
 
 
 
 
 
Mr. Alejandro López-Vázquez (nutritionist) 
 
 
 
 
 
Mr. Juan J. Martin-Olmedo (nutritionist) 
 
 
 
 
 
Mr. Marcos Molina-Fernández (sports scientist) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Name of person writing SAP:  
 
E-mail principal investigator: ruizj@ugr.es 
 
E-mails 
trial 
coordinators: 
acamachocardenos@ugr.es 
& 
claveroa@ugr.es  
PROFITH research group, Department of Physical Education and 
Sports, Sport and Health University Research Institute (iMUDS), 
University of Granada 
 
 
 
 
Signatures: 
 
Prof. Dr. Jonatan R. Ruiz (principal investigator) 
Date: 01-07-2023 
Dr. Alba Camacho-Cardenosa (trial coordinator) 
Date: 01-07-2023 
Mr. Antonio Clavero-Jimeno (trial coordinator) 
Date: 01-07-2023 

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Table of contents 
1. 
Background and rationale .................................................................................................... 3 
2. 
Aims ....................................................................................................................................... 4 
3. 
Methods/Design ..................................................................................................................... 4 
3.1. 
Study design ............................................................................................................................... 4 
3.2. 
Participants and eligibility criteria .......................................................................................... 4 
3.3. 
Recruitment and screening ....................................................................................................... 6 
3.4. 
Outcome measures .................................................................................................................... 6 
3.5. 
Primary outcome: hepatic fat .................................................................................................. 7 
3.6. 
Secondary outcomes .................................................................................................................. 8 
3.7. 
Intervention description ......................................................................................................... 10 
3.8. 
Participant retention, adherence and sustainability ............................................................ 13 
3.9. 
Adverse events ......................................................................................................................... 13 
3.10. 
Data management ................................................................................................................ 13 
3.11. 
Patient and public involvement .......................................................................................... 14 
4. 
Statistical Analysis Plan ...................................................................................................... 14 
4.1. 
Randomization and blinding .................................................................................................. 14 
4.2. 
Sample size ............................................................................................................................... 14 
4.3. 
Statistical analysis ................................................................................................................... 15 
5. 
Ethics and dissemination .................................................................................................... 15 
6. 
References ........................................................................................................................... 15 
 

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1. Background and rationale 
Obesity prevalence has steadily increased up to reach epidemic proportions and affecting 
around 603.7 million adults worldwide (1). The excess of triglycerides in the body is usually 
stored, apart from the subcutaneous adipose tissue (SAT), in other organs and tissues that are 
not otherwise designed for adipose storage (2). This process is known as ectopic fat 
deposition and may include organs and tissues such as the liver, pancreas, or skeletal muscle. 
Excessive accumulation of triglycerides in hepatocytes results in hepatic steatosis, a condition 
considered one of the diagnostic criteria (along with other metabolic dysregulatory factors) 
for metabolic dysfunction-associated steatotic liver disease (MASLD) (3,4). MASLD (which 
replaces non-alcoholic fatty liver disease - NAFLD) is a major public health problem 
considering its elevated prevalence (nearly 90% of adults with overweight/obesity) and its 
strong association with extrahepatic diseases (3,5). Therefore, implementing strategies to 
reduce hepatic steatosis in individuals with obesity may be a potential approach to 
mitigate/reduce the risk of liver dysfunction and cardiometabolic diseases (6). 
Traditionally, low-calorie diets have been shown to be an effective strategy to reduce body 
weight and hepatic steatosis and, in turn, improve cardiometabolic health (7). However, 
energy-restricted approaches are still not a standard public health strategy due to their lack of 
long-term sustainability and some undesirable metabolic adaptations that certainly lead to 
weight regain even in highly motivated patients (8). Time-restricted eating (TRE) is a recently 
emerged intermittent fasting approach which has the potential to maximize the extensively 
reported beneficial metabolic effects of the energy intake restriction (9). TRE aims to 
maintain a consistent daily cycle of feeding (within a limited time window during ≤10 hours) 
and fasting (≥14 hours) to support healthy/consistent circadian rhythms (10). Irregular eating 
patterns and eating over an extended period of time may disrupt circadian rhythms and, thus, 
increase the risk of obesity and hepatic fat accumulation (11). Remarkably, recent studies in 
mice have concluded that TRE effectively reduces hepatic steatosis and improves 
cardiometabolic health (12), mainly through improved insulin sensitivity; yet, whether this 
strategy is similarly effective in humans remains still unclear.   
Along with nutritional strategies, exercise has demonstrated its efficacy at reducing hepatic 
steatosis and at improving cardiometabolic health in humans (13–15). Furthermore, 
preliminary evidence has highlighted that the combination of TRE and exercise may 
normalize glucose homeostasis and improve lipid profile in women with overweight or 
obesity (16). Nevertheless, the differential effects of TRE combined with exercise and TRE 
or exercise alone on hepatic steatosis and cardiometabolic markers remain unknown.  
Although promising, most preliminary pilot trials examining the effects of TRE combined 
with exercise in humans have important limitations: (i) the duration is shorter than 3 months 
(16–19) which may be insufficient to induce substantial changes in cardiometabolic health 
(20); (ii) the outcomes assessed were either body mass index (BMI) as a surrogate marker of 
obesity or bioelectrical impedance analysis which are not able to accurately assess hepatic or 
other ectopic fat depots (18); (iii) the majority of clinical trials have been focused solely on 
men (17–19), or have included an unevenness sample of men and women which limits the 
possibility to understand the important sex dimorphism in MASLD development; (iv) the 
published studies are limited to trained individuals (17–19) and have small sample size and, 
thus, have limited statistical power; (v) the previous work do not include follow-up of the 
participants to understand the maintenance of intervention effects; and, more importantly, (vi) 
have not studied or reported potential mechanisms through which TRE combined with 
exercise may result in health benefits. Therefore, as all these shortcomings limit generality 
and preclude to establish firm conclusions, a new approach is required to successfully 
translate findings into the community and clinical setting.  
 

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2. Aims 
The overall aim of the TEMPUS randomized controlled trial is to investigate the effects of a 
12-week TRE combined with a supervised exercise intervention, as compared with TRE or 
supervised exercise alone, and a usual care control group (UC), on hepatic fat (primary 
outcome) and cardiometabolic health (secondary outcomes) in men and women with obesity.  
3. Methods/Design 
3.1. Study design 
The TEMPUS study is a randomized controlled trial (ClinicalTrials.gov under the identifier 
NCT05897073) with a four-arm parallel design. The protocol of the randomized trial has been 
written and reported based on the Recommendations for Interventional Trials (SPIRIT) 
guidelines (21). Later, the results will be reported following the CONSORT guidelines (REF). 
Consented participants will be randomly assigned to one of the four groups: UC, TRE alone 
(TRE), supervised exercise alone (Exercise), or TRE combined with supervised exercise 
(TRE + Exercise) group. The present study will focus on the recruitment of adults with 
obesity in Granada, a region located in southern Spain. The patient flow diagram from 
recruitment to randomization phases is shown in Figure 1. 
 
Figure 1. TEMPUS project design. TRE, time-restricted eating; CVD, cardiovascular disease. 
3.2. Participants and eligibility criteria 
The study will include both men and women (50%), with a BMI ranging from 30 to <40 
kg/m2, aged between 25 and 65 years, and with a habitual eating window of ³11 hours.  
Detailed criteria for inclusion and exclusion can be found in Table 1. The screening phase, 
as shown in Figure 1, will involve assessing participants' medical history and vital signs to 
determine their eligibility for the study. During the physical examination, any existing 
conditions at the time and any pre-existing medical conditions will be thoroughly 
documented.  
 

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Table 1. Inclusion and exclusion criteria 
Inclusion criteria 
• 
Aged 25-65 years. 
• 
Body mass index between 30 and <40 kg/m2. 
• 
Weight stability (within 3% of screening weight) for >2 months prior to study entry. 
• 
Habitual eating window ≥11 hours.  
Exclusion criteria 
• 
History of a major adverse cardiovascular event (e.g., acute myocardial infarction, ischemic 
or hemorrhagic stroke, or peripheral arterial ischemia, among others), kidney failure, 
chronic 
liver 
disease, 
or 
human 
immunodeficiency virus 
(HIV) 
/ 
acquired 
immunodeficiency syndrome (AIDS).  
• 
Active endocrinological disease, innate errors of metabolism, myopathies or epilepsy.  
• 
Patients who have undergone bariatric surgery or other surgical techniques used for the 
treatment of other pathologies (e.g., "Roux-en-Y"). 
• 
Rheumatoid arthritis, Parkinson's disease, active cancer treatment in the past year or 
another medical condition in which fasting is contraindicated. 
• 
Use of medications that may affect the results of the study such as drugs for glycaemic 
control (e.g., antidiabetic, steroids, beta-blockers, antibiotics, prebiotics, probiotics and 
symbiotics). 
• 
Diagnosis of major sleep or eating disorders.  
• 
Caregiver for a dependent requiring frequent nocturnal care/sleep interruption or shift 
workers with nocturnal hours. 
• 
Metal or electrical prosthesis. 
• 
Foreign bodies in the eyes. 
• 
Fear of needles and claustrophobia to magnetic resonance imaging. 
• 
Active tobacco or illicit drug use or a history of alcohol abuse treatment (i.e., moderate or 
severe alcoholism). 
• 
Participating in a weight loss or a supervised exercise program (i.e., > 30 min in 3 times 
per week, or > 45 min in 2 or more times per week at moderate-to-vigorous intensity).  
• 
Pregnancy and lactation or planned pregnancy (within the study period). 
• 
Frequent travel over time zones during the study period. 
• 
Being unable to understand and to accept the instructions or the study objectives and 
protocol. 
• 
Not having or being able to use a smartphone with Apple iOS or Android OS. 
• 
Are deemed unsuitable by the investigator for any other reason. 

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3.3. Recruitment and screening 
Recruitment of potential participants will be conducted through (i) newspaper advertisements, 
(ii) the Endocrinology and Nutrition Department of the San Cecilio’ and Virgen de las Nieves’ 
University Hospitals of Granada, and (iii) the community of the University of Granada. A pre-
screening process will be implemented with an online form as well as via telephone interviews 
to assess the eligibility of potential participants and engage them in the study. Subsequently, the 
medical team will conduct telephone interviews, to review the patients' medical records and 
evaluate their medical history, verifying compliance with the inclusion and exclusion criteria. 
Potential participants who meet the criteria during the pre-screening phase will be provided with 
oral and written information about the study and will be scheduled for the first evaluation visit. 
During this session, oral and written informed consent will be obtained from potential 
participants, and measurements of body weight and height will be recorded. Additionally, 
participants will perform an incremental exercise test on a treadmill, supervised by the sports 
medicine staff from the Centro Andaluz de Medicina del Deporte and Sport and Health 
University Research Institute, to determine their aptitude for exercising. During the test and at 
rest, electrocardiogram, blood pressure, and capillary blood lactate measurements will be 
obtained. 
3.4. Outcome measures 
A 2-week lead-in period will be implemented prior to pre-intervention assessments and group 
allocation (see Figure 1). During this period, potential participants will be instructed to maintain 
their usual dietary and physical activity habits. They will be provided with a mobile phone 
application (Tempus: com.nnbi.app_extreme_granada; NNBi2020 S.L., Navarra, Spain) to 
record daily information regarding their eating time, sleeping patterns, naps, and any adverse 
events experienced. Additionally, physical activity and sleep quality will be evaluated using 
accelerometry, and glucose excursions over day and night will be assessed using continuous 
glucose monitoring (CGM) devices over the course of 2 weeks.  This data collection will be used 
to verify that the participants' habitual eating windows are ≥11 hours and perform< 30 min in 3 
times per week, or < 45 min in 2 or more times per week at moderate-vigorous intensity activity 
(see inclusion and exclusion criteria, Table 1). Pre-intervention measurements, after 12-week 
intervention measurements (±3 days), and after 12 months post-intervention measurements will 
be conducted by a dedicated team of trained staff to ensure consistency and reliability. In 
addition, we will assess anthropometry and body composition at week 6 (mid-term in Figure 1). 
By including these intermediate measurements, we will potentially capture changes over time 
and evaluate the trajectory of outcomes during the 12-week intervention period. A detailed 
overview of the study's outcomes is shown in Table 2, which provides a comprehensive summary 
of the variables assessed. 
The study primary outcome will be changes from baseline to 12 weeks in hepatic fat. Secondary 
outcome variables include changes from baseline to 12 weeks in other ectopic fat depots (i.e., 
abdominal visceral and subcutaneous adipose tissue, pancreatic fat and mid-thigh intermuscular 
and intramuscular adipose tissue), anthropometry and body composition (i.e., fat mass and fat-
free mass, bone mineral density and content), cardiometabolic markers (i.e., glucose, insulin, 
hemoglobin A1c, lipid profile, vitamin D, alkaline phosphatase and calcium, liver and kidney 
function markers, estimated glomerular filtration rate and steroid and thyroid hormones), 24h 
diurnal and nocturnal mean glucose (via CGM), fecal microbiota, sleep and physical activity 
patterns, psychological outcomes (i.e., depression, stress and anxiety), quality of life, dietary 
habits and eating behaviour assessment, and fitness (i.e. cardiorespiratory fitness and muscular 
strength). After the screening examination and the 2-week lead-in period, pre-intervention 
assessments will be held within 3 face-to-face sessions: 

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Day 1: participants will perform physical fitness tests and will be required to complete 
several web-based questionnaires. These questionnaires will gather information on several 
outcomes such as quality of life, sleeping patterns, psychological status and eating behaviour. 
Dietary intake will be also assessed by qualified nutritionists where participants will be asked to 
provide the first 24-hour recall detailing their food consumption.  
 Day 2: fasting venous blood samples will be collected at the San Cecilio University 
Hospital (Granada, Spain). Thereafter, anthropometric and body composition measurements will 
be conducted at the Sport and  Health University Research Institute (iMUDS - 300 meters apart). 
Finally, oral glucose tolerance tests will be also conducted as part of the glycaemic assessment.   
Day 3: magnetic resonance imaging (MRI) and elastography will be performed in the San 
Cecilio University Hospital by the TEMPUS’ medical staff (Granada, Spain). To ensure 
consistency and standardization during the assessment, participants will be instructed to maintain 
a fasting period of 8-10 hours prior to the appointment, during which they will be advised to 
consume only water and abstain from solid food or other beverages. In addition, participants will 
be advised not to consume alcohol or diuretics 24 hours before the test and to avoid stimulants 
like caffeine for 12 hours before the test. 
 
3.5. Primary outcome: hepatic fat  
The quantification of hepatic fat and iron content will be performed using MRI with a Siemens 
3T Magnetom Vida scanner located at San Cecilio University Hospital in Granada. Additionally, 
liver steatosis, viscosity and fibrosis severity will be assessed using attenuation imaging 
coefficient, shear wave elastography and shear wave dispersion with a Canon Aplio i800 (located 
at San Cecilio University Hospital in Granada). The Fibrosis-4 (FIB-4) index will be calculated 
as an indicator of liver fibrosis severity (22). 
Table 2. Overview of the study outcomes. 
Outcomes 
Pre-
Intervention 
Mid-term 
Post-
Intervention 
12-month 
follow-up 
Primary outcome 
 
 
 
 
Hepatic fat  
a 
 
a 
a 
Secondary outcomes 
 
 
 
 
Ectopic fat depots 
a 
 
a 
a 
Anthropometry and body composition 
a 
a 
a 
a 
Cardiometabolic markers 
a 
 
a 
a 
Glycaemic control 
a 
 
a* 
 
Fecal microbiota 
a 
 
a* 
 
Plasma and fecal metabolomics 
a 
 
a 
 
Sleep and physical activity 
a 
 
a* 
a 
Psychosocial  
a 
 
a 
 
Quality of life 
a 
 
a 
a 
Eating behavior and dietary habits   
a 
 
a 
a 
Fitness 
a 
 
a 
 
*Assessment will be conducted during the last 2 weeks of the intervention.  

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3.6. Secondary outcomes 
Ectopic fat depots: abdominal subcutaneous, visceral and intermuscular adipose tissue, as well 
as pancreatic fat content, will be obtained using MRI. To assess subcutaneous, visceral and 
intermuscular adipose tissue in all 3D abdominal volume (i.e., volume, cross-sectional area at 
selected levels, and mean/median fat fraction), a semiautomatic software program will be 
employed for tissue segmentation. These valuable image markers will be derived from a standard 
6 echo Dixon series, ensuring accurate characterization of abdominal adipose tissue distribution 
and composition. We will also measure image markers from mid-thigh using a 6 echoes Dixon 
series, and will obtain cross-sectional area, muscular tissue, subcutaneous adipose tissue, 
intramuscular adipose tissue, intermuscular adipose tissue, fat fraction, and bone marrow fat 
fraction. The segmentation for all these structures will be performed with a semiautomatic 
proprietary algorithm. 
Anthropometry and body composition: body weight and height measurements will be obtained 
using a stadiometer and scale (Seca model 799, Electronic Column Scale, Hamburg, Germany). 
Participants will be instructed to be barefoot and wear lightweight clothing during these 
measurements. Neck, waist, and hip circumferences, as well as calf girth, will be determined 
following the procedures outlined by the International Society for the Advancement of 
Kinanthropometry (ISAK) by certified personnel (ID’s ISAK: #637686045477240182 and 
#638227824311214767)  (23). Furthermore, fat mass, fat free mass, and overall adipose mass 
will be assessed by bioelectrical bioimpedance (Tanita MC 980-MA Plus, Tanita Corporation, 
Tokyo, Japan), with measurements taken after voiding the bladder to ensure accurate readings. 
Bone mineral density and content will be assessed by dual-energy X-ray absorptiometry scans 
(Discovery Wi, Hologic, Inc., Bedford, MA, USA). Participants will be instructed to maintain a 
fasting period of 8-10 hours prior to the appointment, during which they will be advised to 
consume only water and abstain from solid food or other beverages. In addition, participants will 
be advised to abstain from alcohol or diuretics 24 hours before the test and stimulants like 
caffeine for 12 hours before the test, and avoid engaging in moderate exercise or physical activity 
for 24 hours, or vigorous exercise for 48 hours, prior to the test.  
Cardiometabolic risk markers: venous blood samples will be carefully preserved at a 
temperature of -80°C to ensure their integrity for subsequent analysis. We will collect a full set 
of cardiometabolic risk markers, as these parameters may inform about the metabolic 
dysregulation in liver disease (24). These set will include: fasting glucose (Alinity C system 
analyzer, Abbott Laboratories, IL, USA), insulin (UniCel DxI 800 access immunoassay system, 
Beckman Coulter Inc., CA, USA), hemoglobin A1c (automated glycohemoglobin G11 analyser, 
Horiba) and lipid profile (i.e., total cholesterol, high-density lipoprotein cholesterol, 
triglycerides, apolipoprotein A1 and B) using Alinity C system analyzer (Abbott Laboratories, 
IL, USA). Low-density lipoprotein cholesterol will be calculated using a previously validated 
equation (LDL-c = CT – HDL-c – (TG/5)) (25).  Furthermore, we will measure vitamin D, 
alkaline phosphatase and calcium (Alinity C system analyzer, Abbott Laboratories, IL, USA). 
Liver and kidney function markers (i.e., alanine transaminase, gamma-glutamyl transferase, 
bilirubin, creatinine) will be also measured using an Alinity C system analyzer, while estimated 
glomerular filtration rate, steroid hormones (i.e., estradiol, progesterone, testosterone, follicle 
stimulating hormone and luteinizing) and thyroid hormones (i.e., thyrotropin, thyroxine, 
triiodothyronine) will be also assessed using a UniCel DxI 800 access immunoassay system 
(Beckman Coulter Inc., CA, USA). Blood count and biochemistry and inflammatory markers 
(i.e., iron, ferritin, folic acid, C-reactive protein and interleukin-6) will be measured using a 
AU5800 automated analyzer (Beckman Coulter Inc., CA, USA). Additionally, we will calculate 
insulin resistance surrogates, such as the homeostatic model of assessment for insulin resistance 
(HOMA-IR) and the quantitative insulin-sensitivity check index (QUICKI). Finally, we will also 

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conduct metabolomics analysis on plasma samples. The omics analyses will allow the 
identification of: (i) metabolites strongly associated to hepatic fat content (baseline – cross-
sectional analysis); (ii) metabolites predictors of hepatic fat content changes after the 
intervention. In addition, systolic and diastolic blood pressure will be measured using an 
automated monitor (M3-Comfort, Omron Healthcare Europe B.V., Hoofddorp, The 
Netherlands), following the established guidelines outlined by the 2021 European Society of 
Hypertension (26). 
Glycaemic control: due to the crucial role of insulin resistance in MASLD development, through 
fatty acid accumulation in hepatocytes, CGM and analysis of acute response to glucose will be 
conducted. Participants will be instructed to wear a CGM device (FreeStyle LibrePro, Abbott 
Laboratories, Abbott Park, IL) during 2 weeks before the intervention (lead-in period) and during 
the last 2 weeks of the intervention (weeks 11 and 12). The CGM data obtained will be used to 
calculate different variables related to glycaemic control (i.e., 24-hour mean glucose), following 
the guidelines outlined in the most recent international consensus statement (27). Additionally, 
oral glucose tolerance tests will also be conducted as part of the glycaemic assessment using a 
75-g oral glucose dose (NUTER TEC: orange flavour, Toulouse, France). 
Fecal microbiota: for the comprehensive identification and quantification of fecal microbiome 
diversity and composition, pre-intervention and during the last 2 weeks of the intervention, fecal 
samples will be collected. Stool microbial DNA will be isolated from participants and 
subsequently, 16S rRNA gene amplicon sequencing, with the possibility of employing shotgun 
methodology pending final budget considerations, will be performed. Furthermore, a fecal 
metabolomic fingerprint analysis will be conducted to determine the metabolic profile among 
different groups of patients.  
Sleep and physical activity: sleep quality and chronotype will be evaluated through the 
administration of validated questionnaires, including the Pittsburgh Sleep Quality Index (PSQI) 
(28), Munich Chronotype Questionnaire (MCTQ) (29), and Horne and Östberg Questionnaire for 
Morning-Evening type assessment (MEQ-SA) (30). Objective measures of sleep and physical 
activity levels will be obtained using triaxial accelerometer (ActiGraph GT3X+, Pensacola, FL, 
USA) that participants will wear on their non-dominant wrist during two-week periods: before 
the beginning of the intervention (lead-in period) and during the last two weeks of the 
intervention (weeks 11 and 12).  
Psychosocial assessment: as weight loss intervention may improve emotional well-being and 
psychosocial functioning, several key psychological dimensions will be evaluated through the 
administration of validated questionnaires: the Beck Depression Inventory Fast Screen for 
depression (BDI-FS) (31), the Perceived Stress Scale for stress (PSS) (32), and the State-Trait 
Anxiety Inventory for anxiety (STAI) (33). 
Quality of life: quality of life may also improve from weight loss intervention (34) and will be 
evaluated using the EuroQol 5 dimensions 5 levels (EQ-5D-5L) questionnaire (35) and Rand 
Short Form 36 (SF-36) (36). In addition, participants will be asked to complete an adverse events 
questionnaire to identify any potential adverse effects or complications experienced during the 
intervention.  
Eating behavior and dietary habits: to assess participants' adherence to the Mediterranean 
dietary pattern, validated questionnaires such as the PREDIMED questionnaire will be 
administered. This questionnaire provides a reliable measure of adherence to the specific dietary 
components and guidelines of the Mediterranean diet (37). Food Frequency Questionnaire (FFQ) 
(38) will be administered to assess the frequency that participants have consumed each specific 

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food during the previous 4 weeks. Food Craving Inventory (FCI) (39) and the Adult Eating 
Behavior Questionnaire (AEBQ) for appetite traits (40) will be also administered. 
Physical fitness: an incremental treadmill exercise test until exhaustion will be performed to 
determine cardiorespiratory fitness. The modified Balke protocol (41) will be applied, which has 
been extensively used and validated (42–44). The exercise electrocardiogram and heart rate (HR) 
will be monitored continuously and reviewed by a cardiologist. Furthermore, capillary blood 
lactate will be measured at rest and along different stages of the test (Lactate Pro™ 2 LT-1730, 
Arkray, Kyoto, Japan). Supervised exercise proposed in the present study could restore or 
mitigate adverse effects of diet-induced weight loss on muscle strength (45). Thus, upper 
muscular strength will be assessed through hand grip strength test (46) using a digital hand 
dynamometer (TKK 5401 Grip-D; Takei, Tokyo, Japan), whereas lower body muscular strength 
will be assessed through the 30-s sit-to-stand muscle power test (47), and walking speed to assess 
functional capacity with gait speed test (48).  
Confounding: as unintentional reductions in energy intake (10-30% or ~300-500 kcal/day) have 
been reported when participants confine their eating windows to 4-10 h/day (49,50), we will 
control and analyze changes in energy intake over the intervention period. Participants will 
undergo dietary assessments through the completion of 3 non-consecutive 24-hour dietary recalls 
(2 working days and 1 non-working day) (51). These recalls will be conducted via face-to-face 
or telephone interviews by qualified and trained research nutritionists. Total energy intake, 
carbohydrates, fat and protein intake will be calculated. In addition, to understand the important 
sex dimorphism in MASLD development, at baseline and after the intervention, specific 
reproductive-profile questions will be asked regarding detailed information on menstrual cycle 
history and hormonal contraceptive use and type, as well as regarding any gynecological 
condition. This will help in categorizing participants into different hormonal profiles, following 
the recent consensus (52–54). 
3.7. Intervention description 
3.7.1. Usual care group 
Participants randomly assigned to the UC group will be indicated to continue with their dietary 
eating time schedule. All participants will receive monthly in-person nutritional education 
sessions, lasting approximately 90 minutes, throughout the intervention for weight management 
and cardiovascular health promotion based on Mediterranean dietary patterns (55) and physical 
activity recommendations from the World Health Organization (56). Key points in nutritional 
counseling will be: a) to correctly interpret food labels and to plan grocery, b) to recognize and 
include high satiety foods, and c) to fight against some extended myths in Spanish culture such 
as “alcohol in small quantities is healthy”, “it is unhealthy to skip breakfast”, or “it is important 
to eat 5 meals per day”.  
3.7.2. Time-restricted eating intervention 
Before the beginning of the intervention, participants allocated to TRE groups will select their 
preferred 8-hour eating window before the intervention and will be required to maintain the same 
8-hour eating window during the 12-week intervention. They will be advised that the last meal 
should be completed before or at 21:00. Participants assigned to the TRE groups will be strictly 
prohibited from consuming any calorie-containing food or beverage outside their designated 8-
hour eating window. However, they will be allowed to consume water, coffee, and tea without 
sugar or artificial sweeteners during the fasting period. Instructions for the TRE intervention will 
emphasize its daily implementation, meaning participants are expected to adhere to the eating 
window restriction (±30 minutes) throughout all seven days of the week. 

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3.7.3. Supervised exercise intervention 
The objective of the supervised exercise program will be to ensure its transferability and 
feasibility for the target population. We will follow the Consensus on Exercise Reporting 
Template to facilitate replicability and transparency (57) and record the timing when participants 
are training (58). Thus, the physical activity recommendations for adults proposed by the World 
Health Organization serve as the foundation for determining the specific exercise dosage in the 
TEMPUS study (59). Given that both resistance and aerobic training modalities have shown 
improvements in hepatic steatosis among patients with MASLD (60,61), the TEMPUS study will 
combine both supervised resistance and aerobic high-intensity interval training (HIIT) sessions. 
Qualified sports scientists from outside the research group will carefully supervise the exercise 
sessions and work with groups of no more than six people to ensure that participants perform the 
exercise technique correctly, and at proper intensity. Moreover, participants will receive an 
individualized moderate-intensity goal-setting aerobic (i.e., walking) program consisting of 
increasing 10% daily steps per week based on their daily steps logs. Goal-setting aerobic will be 
updated weekly using a smart band (Xiaomi Mi Band 7, Xiaomi, Pekin, China) that participants 
enrolled in the exercise groups will wear on their wrists during the intervention period.  
Volume: participants will engage in a 12-week intervention program including 2 supervised 
exercise sessions per week ranging from 60 to 90 minutes per session. Furthermore, participants 
will receive instructions to complete their personalized daily step goals established for each day.  
Intensity: supervised sessions will start with a supervised circuit-based resistance training that 
focuses on upper and lower body exercises that target major muscle groups. To prescribe 
intensity, the rate of perceived exertion (RPE) scale ranging from 0 to 10 will be utilized, using 
the evaluations and first sessions to properly train the participants on this scale (62). The target 
intensity for resistance training will be set at an RPE level greater than 7, which will vary 
depending on the week number of the intervention (from 7 to 10). The participants will be 
encouraged to achieve this intensity through all the exercises performed in each lap of the circuit. 
Moreover, training sessions will include HIIT as the vigorous-intensity aerobic exercise 
component. HIIT will consist of 3-4 sets of 4-minute intervals at >85% of the individual heart 
rate reserve (HRR) with 4 minutes of active recovery at 65-75% HRR. HRR will be calculated 
considering the peak of HR achieved in the incremental treadmill test and the rest HR lying in 
bed recorded during pre-intervention assessments. HR will be continuously monitored during all 
exercise sessions using the Polar H10 band (Polar Products Inc., Stow, OH, USA). 
Frequency: participants will perform 2 supervised exercise training sessions per week, with a 
resting period of at least 48 hours between them. Additionally, participants will be instructed to 
accomplish the prescribed daily step goal. If a participant misses a training session, it will be 
rescheduled and recovered considering a minimum resting period of 48 hours between sessions. 
Type of exercise: the resistance training sessions will be composed of major upper and lower 
body muscle groups including (63): horizontal/vertical pull exercises (i.e., seated low row and 
lat pulldown with resistance elastic bands), hip-dominant exercises (i.e., deadlift with resistance 
elastic bands and weight-bearing glute bridge), knee-dominant exercises (i.e., weight-bearing 
squats and lunges), and horizontal/vertical push exercises (i.e., weight-bearing push-ups and 
shoulder press with resistance elastic bands). We will propose 4 levels of exercises’ difficulty 
based on the resistance elastic band and the progressive complexity of the basic movement 
patterns involved. After the resistance circuit training, HIIT will be preferably performed on a 
treadmill to ensure a suitable progressive overload. As alternative modality, elliptical ergometer 
will be used. 

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Training load variation: we are aware that participants might not be immediately capable of 
exercising at high intensities and volumes. Therefore, there will be a gradual progression to the 
assigned exercise dose in 3 phases (see Figure 2) and proper technique will be a priority for the 
difficulty level progression to avoid potential injuries. 
Training periodization: the supervised exercise intervention will be divided into 3 phases (Figure 
2). The initial phase will have a length of 6 weeks and will start with a first week of 
familiarization. During this week, participants will learn the structure and organization of the 
sessions and the movement patterns that constitute the base of the different exercises. 
Considering that an inactive person may be unable to immediately train at the selected doses, the 
familiarization phase will prepare participants to gradually increase the workload until the 
required dose is achieved. On the other hand, the resistance training will set light loads using 
resistance elastic bands or weight-bearing exercises. They will increase the load and coordinative 
difficulty (through 4 levels for each exercise) as soon as participants perform the exercises with 
a proper technique.  
We expect a parallel increase in the intensity of both resistance training and HIIT as participants' 
fitness levels improve. Specifically, adjustments (i.e., greater speed or power) to HIIT will be 
controlled based on the HRR. This approach is essential to maintain a targeted percentage of 
HRR, particularly as participants exhibit enhanced fitness capacities. 
Training session: a training session will be organized as follow: (i) a warm-up of one circuit 
training lap with light loads using resistance elastic bands or weight-bearing exercises (20 
seconds work: 20 seconds rest), (ii) 2 to 4 circuit training laps of eight exercises (30 seconds 
work: 30 seconds rest) at 7-10 RPE, with a between-set rest of 3-5 min, and (iii) 3 or 4 sets of  4-
min intervals at 85-95% HRR interposed by 4 min of active recovery at 65-75% of HRR, with 2 
min prior of warm-up. A cool-down protocol of 5 min will be performed at the end including 
anterior and/or posterior chain exercises for muscle elongation and/or relaxation. The total 
duration of exercise sessions will be 55 minutes in the familiarization week, 65 minutes during 
the first phase, 75 minutes in the second phase, and 85 minutes in the third phase.  
 
Figure 2. Supervised exercise periodization of the TEMPUS project. *Week of familiarization. HIIT, high-intensity 
interval training; HRR, heart rate reserve; RPE, rate of perceived exertion; RT, resistance training. AET, aerobic 
endurance training. 
 
Phase 
FIRST 
SECOND 
THIRD 
Week 
1* 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
Supervised Exercise Volume (min/week) 
110 
130 
130 
130 
130 
130 
150 
150 
150 
150 
170 
170 
Warm-up (RT) 
Volume (min/session) 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
Resistance training 
Total volume (min/week) 
32 
32 
32 
32 
32 
32 
48 
48 
48 
48 
64 
64 
Intensity  
(RPE 1-10 scale) 
7 
7 - 8 
7 - 8 
7 - 8 
8 - 9 
8 - 9 
8 - 9 
8 - 9 
9 - 10 
9 - 10 
9 - 10 
9 - 10 
Secs work/Secs rest 
(per exercise) 
30/30 
30/30 
30/30 
30/30 
30/30 
30/30 
30/30 
30/30 
30/30 
30/30 
30/30 
30/30 
Number of laps 
(per session) 
2 
2 
2 
2 
2 
2 
3 
3 
3 
3 
4 
4 
Rest between laps (min) 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
Number of exercises  
(per session) 
8 
8 
8 
8 
8 
8 
8 
8 
8 
8 
8 
8 
Rest between 
RT & AET 
Volume (min/session) 
3 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
Warm-up (HIIT) 
Volume (min/session) 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
HIIT 
Vigorous-intensity 
intervals / Moderate-
intensity intervals 
3/3 
4/4 
4/4 
4/4 
4/4 
4/4 
4/4 
4/4 
4/4 
4/4 
4/4 
4/4 
Total volume vigorous-
intensity (min/week) 
24 
32 
32 
32 
32 
32 
32 
32 
32 
32 
32 
32 
Intensity (%HRR) 
80 - 85 
85 - 95 
85 - 95 
85 - 95 
85 - 95 
85 - 95 
85 - 95 
85 - 95 
85 - 95 
85 - 95 
85 - 95 
85 - 95 
Total volume moderate-
intensity (min/week) 
24 
32 
32 
32 
32 
32 
32 
32 
32 
32 
32 
32 
Intensity (%HRR) 
60 - 65 
65 - 75 
65 - 75 
65 - 75 
65 - 75 
65 - 75 
65 - 75 
65 - 75 
65 - 75 
65 - 75 
65 - 75 
65 - 75 
Cool down 
Volume (min/session) 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 

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3.8. Participant retention, adherence and sustainability 
The Principal Investigator and study team will exert every effort to facilitate participants' 
completion of all study visits and ensure overall study retention. The following strategies will be 
implemented to maximize participants' retention and minimize loss to follow-up: a) 
implementing a proactive retention plan that focuses on building close participant relations and 
ensuring participant satisfaction, b) giving the opportunities for participants and their families to 
ask questions and voice any concerns related to their condition throughout the study, c) 
reinforcing comprehension of the objectives and protocol of the study during study visits or 
conducting question and answer sessions after each visit, and d) evaluating each likelihood of 
dropout and implementing appropriate interventions to maintain their interest and motivation to 
continue participating in the study. 
All supervised exercise sessions will be performed in a well-lit and airy room, providing to the 
participants the opportunity to choose their own music. The training specialists and other study 
staff will consistently offer support to participants throughout the duration of the study. 
During the 12-week intervention period, participants will be required to record their daily sleep 
and eating times (i.e., exact times of the beginning of the first meal and of the end of the last 
meal), as well as any potential adverse events in a mobile phone app specifically designed for the 
study. These data will be revised 2 to 3 times every week, asking the participant for missing 
records, and will provide insights into their adherence to the prescribed eating window. 
Participants in the exercise groups and TRE groups will be labeled as “adherent” if they perform 
>80% of training sessions and eat within their allocated window of 8h (±30 minutes) >80% of 
days. Finally, we will assess the long-term adherence to the intervention (at the 12-month follow-
up). This will allow us to evaluate the sustainability of participants' adherence over an extended 
period.  
3.9.  Adverse events 
The study coordinators and project managers will oversee the collection of data and monitor the 
frequency of reported adverse events on a weekly basis using the mobile phone app (Tempus: 
com.nnbi.app_extreme_granada; NNBi2020 S.L., Navarra, Spain). Additionally, participants 
will complete validated questionnaires that assess gastrointestinal and autonomic symptoms, 
well-being, eating behavior, sleep quality, stress levels, mood, anxiety, and depression. These 
questionnaires will offer valuable information on any potential adverse effects and overall health-
related outcomes. If a serious adverse event or an unanticipated problem occurs, the study 
coordinators will immediately notify both the principal investigators and the medical staff. 
Subsequently, a collective decision will be made and, if needed, the ethics committee will be 
properly informed. Moreover, appropriate measures will be taken to address and manage the 
reported event effectively. 
3.10. Data management 
Data collected will be directly entered into REDCap (Research Electronic Data Capture), a secure 
web-based platform specifically designed to create and manage research-related databases and 
surveys. This platform will ensure data security and confidentiality. For any data not recorded in 
REDCap, strict access control measures will be implemented to securely store the data on 
university computers, maintaining confidentiality and data integrity. 
To ensure data quality and integrity, regular quality control checks will be conducted to identify 
any potential data anomalies, such as missing data or forms, data that falls outside the expected 
range, erroneous data entries, illogical dates over time, data inconsistencies across different forms 
and study visits, and incomplete fields on completed forms without a valid explanation for the 

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missing data. Any identified issues will be promptly reviewed and addressed by the data 
monitoring committee to ensure the accuracy and reliability of the data. The data monitoring 
committee will be independent from the sponsor. There are no auditing procedures planned.  
3.11.  Patient and public involvement  
TEMPUS will stimulate patient and public involvement throughout the entire process. Potential 
participants have actually been involved in the preparation of the intervention and in the 
development of the mobile app, organization of the outcome measures, as well as are helping in 
the recruitment of new participants. Once we have the results ready, we will actively involve the 
participants in the reporting and advocacy of the study results. 
4. Statistical Analysis Plan 
4.1. Randomization and blinding 
We expect to recruit and randomize ~184 participants using both stratification and permuted 
blocks with random block sizes, after completing the pre-intervention measurements. 
Randomization will be stratified based on sex (men-women), resulting in 2 strata. Prior to the 
beginning of the trial, randomization lists will be generated for each strata. The block sizes will 
be randomly determined, with allowable sizes of 4 and 8. Within each block, random selection 
will be used to assign participants to one of the four possible groups (i.e., UC, TRE, Exercise or 
TRE + Exercise) using a parallel design with a 1:1:1:1 allocation ratio. The sequential assignment 
of participants will follow the predetermined randomization list specific to their strata. The 
utilization of random block sizes ensures that the next assignment cannot be predicted. 
Stratification by sex ensures that the intervention groups will be balanced for this important 
characteristic. Blinding procedures will be rigorously applied to all personnel responsible for 
assessing primary outcomes, specifically hepatic fat depots and other ectopic fat depots derived 
from MRI. This blinding will also extend to those analyzing cardiometabolic risk factors, 
conducting glucose monitoring, examining fecal microbiota, evaluating physical activity and 
sedentary time, as well as those engaged in the statistical analysis of data. Conversely, personnel 
responsible for other measurements and intervention administration will not be blinded to the 
group assignment (open-label). Participants will receive clear information about the group they 
will be assigned to, along with details about the study hypotheses. In order to ensure practicality 
and feasibility, and based on previous research experience (64–66), the study will be conducted 
in successive waves of participants, each of them including a maximum of 16 participants. 
4.2. Sample size 
Based on previous findings from a recent trial on the combination of alternate day fasting and 
exercise on hepatic fat content (67), we anticipate approximately 5.0% reduction in hepatic fat 
content in the TRE + Exercise group, 2.5% in the TRE group, 2.5% in the exercise group, and 
no significant change in this outcome in the usual-care group. Assuming a pre-post correlation 
of 0.8 and a standard deviation of 6 points in the main outcome, we estimate a medium effect 
size of 0.45. To detect this effect size as statistically significant in a one-way ANOVA with α = 
0.05 and a power of 0.8, a minimum of 19 patients per group is required. Accounting for subgroup 
analyses based on sex and a maximum dropout rate of 20%, we will aim to recruit ~46 
participants for each trial group, resulting in a total sample size of ~184 participants, with ~92 
women included. To ensure a balanced representation of both sexes and maintain an adequate 
sample size, we will implement several strategies: 
- 
Recruitment Process: A specific recruitment process will be utilized to aim for an equal 
enrollment of men and women. For each woman recruited, we will encourage her to invite 
one man to participate in the study. This approach has been successfully employed in our 
previous intervention studies and has proven effective in achieving a balanced sex 

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distribution (68–70). 
- 
Sample size calculations: Our sample size calculations have taken into account subgroup 
analyses by sex. We have conservatively estimated a maximum dropout rate of 
approximately 20%. By considering this dropout rate, we have ensured that our study is 
adequately powered to detect the specified effect size even if there are differential dropout 
rates between men and women. For example, if men have a dropout rate of 5% and 
women have a dropout rate of 15%, our study will still have sufficient power to analyze 
the data separately in men and women. 
- 
Expected dropout rate: While we have conservatively estimated the maximum dropout 
rate, we anticipate that the actual dropout rate will be relatively lower and similar between 
both sexes. This expectation is based on our previous studies and the measures we have 
to promote participant engagement and adherence (68–70). 
4.3. Statistical analysis 
The effects on primary and secondary outcomes in response to the present 12-week intervention 
will be assessed based on repeated-measures linear mixed-effects multilevel models. Individual 
measures of change will be therefore modeled as a function of the randomly assigned group, 
assessment time, and their interaction terms. All the analyses will also be conducted separately 
for men and women. Model-based estimations will be performed with an intention-to-treat 
approach (primary analyses) using the restricted maximum-likelihood method, the model 
assuming that missing values are missing-at-random. Analyses and estimations will also be 
performed with a per-protocol approach, and an attrition propensity will be calculated using a 
logistic model predicting attrition with baseline values of allocation group, age, sex, and BMI. 
Additional models will be conducted including energy intake, physical activity, or reproductive 
status in women. In addition to the conventional approach of assessing intervention effects 
based on statistical and practical significance, it is important to highlight that this study will 
employ a practical benefit approach. This approach emphasizes the reporting of unadjusted 
values that are intuitive to human judgment and easily replicable, considering the design and 
methodology of the study.  
The main statistical analyses will be performed by an independent researcher (Dr. Almudena 
Carneiro-Barrera), who is not involved in the recruitment, evaluations, and interventions, and 
will be performed blinded to the treatment allocation by coding the intervention arms (e.g., A, 
B, C, D). 
 
5. Ethics and dissemination 
The study has received ethical approval from the Granada Provincial Research Ethics Committee 
(CEI Granada - 0365-N-23) and will be performed following the ethical guidelines of the 
Declaration of Helsinki. Before their inclusion in the study, participants will be required to 
provide oral and written informed consent, with further details available in the Online Supporting 
Information. 
Results will be presented in peer-reviewed, scientific journals and at international conferences. 
We aim to publish a main paper with the primary outcome data. Since it is a large study with 
numerous secondary outcomes, other specific manuscripts on each topic will be published. 
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extension power and 30-s sit-to-stand muscle power in older adults: validation and translation to 
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weight loss: progress and future directions. Nat Rev Endocrinol. 2022;18(5):309–21.  
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Ruiz JR, Sevilla‐Lorente R, Amaro‐Gahete FJ. Time for precision exercise prescription: the same 
timing may not fit all. J Physiol [Internet]. 2023 Dec 13 [cited 2023 Dec 14]; Available from: 
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Organization 2020 guidelines on physical activity and sedentary behaviour. Br J Sports Med 
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exercise in non-alcoholic fatty liver disease: A systematic review. J Hepatol [Internet]. 2017 Jan 1 
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61. 
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64. 
Martinez-Tellez B, Sanchez-Delgado G, Acosta FM, Alcantara JMA, Amaro-Gahete FJ, Martinez-
Avila WD, et al. No evidence of brown adipose tissue activation after 24 weeks of supervised 
exercise training in young sedentary adults in the ACTIBATE randomized controlled trial. Nat 
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65. 
Carneiro-Barrera A, Amaro-Gahete FJ, Guillén-Riquelme A, Jurado-Fasoli L, Sáez-Roca G, 
Martín-Carrasco C, et al. Effect of an Interdisciplinary Weight Loss and Lifestyle Intervention on 
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66. 
Amaro-Gahete FJ, De-la-O A, Jurado-Fasoli L, Dote-Montero M, Gutiérrez Á, Ruiz JR, et al. 
Changes in Physical Fitness After 12 Weeks of Structured Concurrent Exercise Training, High 
Intensity Interval Training, or Whole-Body Electromyostimulation Training in Sedentary Middle-
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Clinical and Translational Report Effect of alternate day fasting combined with aerobic exercise 
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combined with aerobic exercise on non-alcoholic fatty liv. Cell Metab. 2023;1–15.  
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Martinez-Tellez B, Sanchez-Delgado G, Acosta FM, Alcantara JMA, Amaro-Gahete FJ, Martinez-
Avila WD, et al. No evidence of brown adipose tissue activation after 24 weeks of supervised 
exercise training in young sedentary adults in the ACTIBATE randomized controlled trial. Nat 
Commun. 2022 Sep 12;13(1):5259.  
69. 
Amaro-Gahete FJ, De-la-O A, Jurado-Fasoli L, Dote-Montero M, Gutiérrez Á, Ruiz JR, et al. 
Changes in Physical Fitness After 12 Weeks of Structured Concurrent Exercise Training, High 
Intensity Interval Training, or Whole-Body Electromyostimulation Training in Sedentary Middle-
Aged Adults: A Randomized Controlled Trial. Front Physiol. 2019;10:451.  
70. 
Labayen I, Medrano M, Arenaza L, Maíz E, Osés M, Martínez-Vizcaíno V, et al. Effects of 
Exercise in Addition to a Family-Based Lifestyle Intervention Program on Hepatic Fat in Children 
With Overweight. Diabetes Care. 2020 Feb;43(2):306–13.  
  
 

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TEMPUS 
 
 
 
 
 
Study Protocol 
 
 
 
 
 
 
University of Granada, Spain 
Trial registration number: NCT05897073 
Version: 2 

TEMPUS, Study Protocol 
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22 
Date: 01-07-2024 
 
 
 
 
Roles and responsibility: 
Name and address of principle 
Prof. Dr. Jonatan R. Ruiz (principal investigator), 
investigator and trial coordinators: 
Dr. Alba Camacho-Cardenosa (trial coordinator) 
Mr. Antonio Clavero-Jimeno (trial coordinator) 
PROFITH (PROmoting FITness and Health Through Physical 
Activity) research group, Department of Physical Education and 
Sports, Sport and Health University Research Institute (iMUDS), 
University of Granada 
Faculty of Sport Science, Carretera de Alfacar s/n, Granada, 
18071, Spain 
 
Study Team 
 
 
 
Dr. Adrián Cortés-Martín (biochemist) 
 
 
 
 
 
Dr. Alejandro De-la-O (data manager) 
 
 
 
 
 
Dr. José Luis Martín Rodríguez (radiologist) 
Dr. Manuel Muñoz-Torres (endocrinologist) 
 
 
 
 
 
Ms. Balma Boira-Nacher (nutritionist) 
 
 
 
 
 
Mr. Alejandro López-Vázquez (nutritionist) 
 
 
 
 
 
Mr. Juan J. Martin-Olmedo (nutritionist) 
 
 
 
 
 
Mr. Marcos Molina-Fernández (sports scientist) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Name of person writing SAP:  
 
E-mail principal investigator: ruizj@ugr.es 
 
E-mails 
trial 
coordinators: 
acamachocardenos@ugr.es 
& 
claveroa@ugr.es  
PROFITH research group, Department of Physical Education and 
Sports, Sport and Health University Research Institute (iMUDS), 
University of Granada 
 
 
 
 
Signatures: 
 
Prof. Dr. Jonatan R. Ruiz (principal investigator) 
Date: 01-07-2024 
Dr. Alba Camacho-Cardenosa (trial coordinator) 
Date: 01-07-2024 
Mr. Antonio Clavero-Jimeno (trial coordinator) 
Date: 01-07-2024 

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Table of contents 
1. 
Background and rationale .................................................................................................. 24 
2. 
Aims ..................................................................................................................................... 25 
3. 
Methods/Design ................................................................................................................... 25 
3.1. 
Study design ............................................................................................................................. 25 
3.2. 
Participants and eligibility criteria ........................................................................................ 25 
3.3. 
Recruitment and screening ..................................................................................................... 27 
3.4. 
Outcome measures .................................................................................................................. 27 
3.5. 
Primary outcome: hepatic fat ................................................................................................ 29 
3.6. 
Secondary outcomes ................................................................................................................ 29 
3.7. 
Intervention description ......................................................................................................... 31 
3.8. 
Participant retention, adherence and sustainability ............................................................ 34 
3.9. 
Adverse events ......................................................................................................................... 35 
3.10. 
Data management ................................................................................................................ 35 
3.11. 
Patient and public involvement .......................................................................................... 35 
4. 
Statistical Analysis Plan ...................................................................................................... 35 
4.1. 
Randomization and blinding .................................................................................................. 35 
4.2. 
Sample size ............................................................................................................................... 36 
4.3. 
Statistical analysis ................................................................................................................... 36 
5. 
Ethics and dissemination .................................................................................................... 37 
6. 
References ........................................................................................................................... 37 
7. 
Summary of changes to Study Protocol ............................................................................. 43 
 
 
 

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1. Background and rationale 
Obesity prevalence has steadily increased up to reach epidemic proportions and affecting 
around 603.7 million adults worldwide (1). The excess of triglycerides in the body is usually 
stored, apart from the subcutaneous adipose tissue (SAT), in other organs and tissues that are 
not otherwise designed for adipose storage (2). This process is known as ectopic fat 
deposition and may include organs and tissues such as the liver, pancreas, or skeletal muscle. 
Excessive accumulation of triglycerides in hepatocytes results in hepatic steatosis, a condition 
considered one of the diagnostic criteria (along with other metabolic dysregulatory factors) 
for metabolic dysfunction-associated steatotic liver disease (MASLD) (3,4). MASLD (which 
replaces non-alcoholic fatty liver disease - NAFLD) is a major public health problem 
considering its elevated prevalence (nearly 90% of adults with overweight/obesity) and its 
strong association with extrahepatic diseases (3,5). Therefore, implementing strategies to 
reduce hepatic steatosis in individuals with obesity may be a potential approach to 
mitigate/reduce the risk of liver dysfunction and cardiometabolic diseases (6). 
Traditionally, low-calorie diets have been shown to be an effective strategy to reduce body 
weight and hepatic steatosis and, in turn, improve cardiometabolic health (7). However, 
energy-restricted approaches are still not a standard public health strategy due to their lack of 
long-term sustainability and some undesirable metabolic adaptations that certainly lead to 
weight regain even in highly motivated patients (8). Time-restricted eating (TRE) is a recently 
emerged intermittent fasting approach which has the potential to maximize the extensively 
reported beneficial metabolic effects of the energy intake restriction (9). TRE aims to 
maintain a consistent daily cycle of feeding (within a limited time window during ≤10 hours) 
and fasting (≥14 hours) to support healthy/consistent circadian rhythms (10). Irregular eating 
patterns and eating over an extended period of time may disrupt circadian rhythms and, thus, 
increase the risk of obesity and hepatic fat accumulation (11). Remarkably, recent studies in 
mice have concluded that TRE effectively reduces hepatic steatosis and improves 
cardiometabolic health (12), mainly through improved insulin sensitivity; yet, whether this 
strategy is similarly effective in humans remains still unclear.   
Along with nutritional strategies, exercise has demonstrated its efficacy at reducing hepatic 
steatosis and at improving cardiometabolic health in humans (13–15). Furthermore, 
preliminary evidence has highlighted that the combination of TRE and exercise may 
normalize glucose homeostasis and improve lipid profile in women with overweight or 
obesity (16). Nevertheless, the differential effects of TRE combined with exercise and TRE 
or exercise alone on hepatic steatosis and cardiometabolic markers remain unknown.  
Although promising, most preliminary pilot trials examining the effects of TRE combined 
with exercise in humans have important limitations: (i) the duration is shorter than 3 months 
(16–19) which may be insufficient to induce substantial changes in cardiometabolic health 
(20); (ii) the outcomes assessed were either body mass index (BMI) as a surrogate marker of 
obesity or bioelectrical impedance analysis which are not able to accurately assess hepatic or 
other ectopic fat depots (18); (iii) the majority of clinical trials have been focused solely on 
men (17–19), or have included an unevenness sample of men and women which limits the 
possibility to understand the important sex dimorphism in MASLD development; (iv) the 
published studies are limited to trained individuals (17–19) and have small sample size and, 
thus, have limited statistical power; (v) the previous work do not include follow-up of the 
participants to understand the maintenance of intervention effects; and, more importantly, (vi) 
have not studied or reported potential mechanisms through which TRE combined with 
exercise may result in health benefits. Therefore, as all these shortcomings limit generality 
and preclude to establish firm conclusions, a new approach is required to successfully 
translate findings into the community and clinical setting.  
 

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2. Aims 
The overall aim of the TEMPUS randomized controlled trial is to investigate the effects of a 
12-week TRE combined with a supervised exercise intervention, as compared with TRE or 
supervised exercise alone, and a usual care control group (UC), on hepatic fat (primary 
outcome) and cardiometabolic health (secondary outcomes) in men and women with obesity.  
3. Methods/Design 
3.1. Study design 
The TEMPUS study is a randomized controlled trial (ClinicalTrials.gov under the identifier 
NCT05897073) with a four-arm parallel design. The protocol of the randomized trial has been 
written and reported based on the Recommendations for Interventional Trials (SPIRIT) 
guidelines (21). Later, the results will be reported following the CONSORT guidelines. 
Consented participants will be randomly assigned to one of the four groups: UC, TRE alone 
(TRE), supervised exercise alone (Exercise), or TRE combined with supervised exercise 
(TRE + Exercise) group. The present study will focus on the recruitment of adults with 
obesity in Granada, a region located in the southern Spain. The patient flow diagram from 
recruitment to randomization phases is shown in Figure 1. 
 
Figure 1. TEMPUS project design. TRE, time-restricted eating; CVD, cardiovascular disease. 
3.2. Participants and eligibility criteria 
The study will include both men and women (50%), with a BMI ranging from 30 to <40 
kg/m2, aged between 25 and 65 years, and with a habitual eating window of ³11 hours.  
Detailed criteria for inclusion and exclusion can be found in Table 1. The screening phase, 
as shown in Figure 1, will involve assessing participants' medical history and vital signs to 
determine their eligibility for the study. During the physical examination, any existing 
conditions at the time and any pre-existing medical conditions will be thoroughly 
documented.  
 

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26 
 
Table 1. Inclusion and exclusion criteria 
Inclusion criteria 
• 
Aged 25-65 years. 
• 
Body mass index between 30 and <40 kg/m2. 
• 
Weight stability (within 3% of screening weight) for >2 months prior to study entry. 
• 
Habitual eating window ≥11 hours.  
Exclusion criteria 
• 
History of a major adverse cardiovascular event (e.g., acute myocardial infarction, ischemic 
or hemorrhagic stroke, or peripheral arterial ischemia, among others), kidney failure, 
chronic 
liver 
disease, 
or 
human 
immunodeficiency virus 
(HIV) 
/ 
acquired 
immunodeficiency syndrome (AIDS).  
• 
Active endocrinological disease, innate errors of metabolism, myopathies or epilepsy.  
• 
Patients who have undergone bariatric surgery or other surgical techniques or used for the 
treatment of other pathologies (e.g., "Roux-en-Y”). 
• 
Rheumatoid arthritis, Parkinson’s disease, active cancer treatment in the past year or 
another medical condition in which fasting is contraindicated. 
• 
Use of medications that may affect the results of the study such as drugs for glycaemic 
control (e.g., antidiabetic, steroids, beta-blockers, antibiotics, prebiotics, probiotics and 
symbiotics). 
• 
Diagnosis of major sleep or eating disorders.  
• 
Caregiver for a dependent requiring frequent nocturnal care/sleep interruption or shift 
workers with nocturnal hours. 
• 
Metal or electrical prosthesis. 
• 
Foreign bodies in the eyes. 
• 
Fear of needles and claustrophobia to magnetic resonance imaging. 
• 
Active tobacco or illicit drug use or a history of alcohol abuse treatment (i.e., moderate or 
severe alcoholism). 
• 
Participating in a weight loss or a supervised exercise program (i.e., > 30 min in 3 times 
per week, or > 45 min in 2 or more times per week at moderate-to-vigorous intensity).  
• 
Pregnancy and lactation or planned pregnancy (within the study period). 
• 
Frequent travel over time zones during the study period. 
• 
Being unable to understand and to accept the instructions or the study objectives and 
protocol. 
• 
Not having or being able to use a smartphone with Apple iOS or Android OS. 
• 
Are deemed unsuitable by the investigator for any other reason. 

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3.3. Recruitment and screening 
Recruitment of potential participants will be conducted through (i) newspaper 
advertisements, (ii) the Endocrinology and Nutrition Department of the San Cecilio’ and 
Virgen de las Nieves’ University Hospitals of Granada, and (iii) the community of the 
University of Granada. A pre-screening process will be implemented with an online form 
as well as via telephone interviews to assess eligibility of potential participants and 
engage them in the study. Subsequently, the medical team will conduct telephone 
interviews, to review the patients’ medical records and evaluate their medical history, 
verifying compliance with the inclusion and exclusion criteria. Potential participants who 
meet the criteria during the pre-screening phase will be provided with oral and written 
information about the study and will be scheduled for the first evaluation visit. During 
this session, oral and written informed consent will be obtained from potential 
participants, and measurements of body weight and height will be recorded. Additionally, 
participants will perform an incremental exercise test on a treadmill, supervised by the 
sports medicine staff from the Centro Andaluz de Medicina del Deporte and Sport and 
Health University Research Institute, to determine their aptitude for exercising. During 
the test and at rest, electrocardiogram and blood pressure measurements will be obtained. 
3.4. Outcome measures 
A 2-week lead-in period will be implemented prior to pre-intervention assessments and 
group allocation (see Figure 1). During this period, potential participants will be 
instructed to maintain their usual dietary and physical activity habits. They will be 
provided with a mobile phone application (Tempus: com.nnbi.app_extreme_granada; 
NNBi2020 S.L., Navarra, Spain) to record daily information regarding their eating time, 
sleeping patterns, naps, and any adverse events experienced. Additionally, physical 
activity and sleep quality will be evaluated using accelerometry, and glucose excursions 
over day and night will be assessed using continuous glucose monitoring (CGM) devices 
over the course of 2 weeks.  This data collection will be used to verify that the participants' 
habitual eating windows are ≥11 hours and perform < 30 min in 3 times per week, or < 
45 min in 2 or more times per week at moderate-vigorous intensity activity (see inclusion 
and exclusion criteria, Table 1). Pre-intervention measurements, after 12-week 
intervention measurements (±3 days), and after 12 months post-intervention 
measurements will be conducted by a dedicated team of trained staff to ensure consistency 
and reliability. In addition, we will assess anthropometry and body composition at week 
6 (mid-term in Figure 1). By including these intermediate measurements, we will 
potentially capture changes over time and evaluate the trajectory of outcomes during the 
12-week intervention period. A detailed overview of the study's outcomes in shown in 
Table 2, which provides a comprehensive summary of the variables assessed. 
The study primary outcome will be changes from baseline to 12 weeks in hepatic fat. 
Secondary outcome variables include changes 12 weeks to 12 months in hepatic fat, 
changes from baseline to 12 weeks and change from 12 weeks to 12 months in other 
specific fat depots (i.e., visceral and abdominal subcutaneous adipose tissue, pancreatic 
fat and mid-thigh intermuscular and intramuscular adipose tissue), elastography (i.e., liver 
stiffness, liver steatosis and liver viscosity), anthropometry and body composition (i.e., 
fat mass and fat free mass, bone mineral density and content and skeletal muscle tissue – 
via MRI-), cardiometabolic markers (i.e., fating glucose, fating insulin, hemoglobin A1c 
and the homeostatic model of assessment for insulin resistance [HOMA-IR], lipid profile, 
liver markers, blood pressure and inflammatory biomarkers), 24h, diurnal and nocturnal 
mean glucose (via CGM), fecal microbiota, sleep and physical activity patterns, 

TEMPUS, Study Protocol 
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28 
psychological outcomes (i.e., depression, stress and anxiety), quality of life, dietary habits 
and eating behaviour assessment, and physical fitness (i.e. cardiorespiratory fitness, lower 
muscular strength, upper muscular strength and walking speed). After the screening 
examination and the 2-week lead-in period, pre-intervention assessments will be held 
within 3 face-to-face sessions: 
Day 1: participants will perform physical fitness tests and will be required to 
complete several web-based questionnaires. These questionnaires will gather information 
on several outcomes such as quality of life, sleeping patterns, psychological status and 
eating behaviour. Dietary intake will be also assessed by qualified nutritionist where 
participants will be asked to provide the first 24-hour recall detailing their food 
consumption.  
 Day 2: fasting venous blood samples will be collected at the San Cecilio 
University Hospital (Granada, Spain). Thereafter, anthropometric and body composition 
measurements will be conducted at the Sport and  Health University Research Institute 
(iMUDS - 300 meters apart). Finally, oral glucose tolerance tests will be also conducted 
as part of the glycaemic assessment.   
Day 3: magnetic resonance imaging (MRI) and elastography will be performed in 
the San Cecilio University Hospital by the TEMPUS’ medical staff (Granada, Spain). To 
ensure consistency and standardization during the assessment, participants will be 
instructed to maintain a fasting period of 8-10 hours prior to the appointment, during 
which they will be advised to consume only water and abstain from solid food or other 
beverages. In addition, participants will be advised not to consume alcohol or diuretics 
24 hours before the test and to avoid stimulants like caffeine for 12 hours before the test. 
 
Table 2. Overview of the study outcomes. 
Outcomes 
Pre-
Intervention Mid-term 
Post-
Intervention 
12-month 
follow-up 
Primary outcome 
 
 
 
 
Hepatic fat  
a 
 
a 
a 
Secondary outcomes 
 
 
 
 
Ectopic fat depots 
a 
 
a 
a 
Anthropometry and body 
composition 
a 
a 
a 
a 
Cardiometabolic markers 
a 
 
a 
a 
Glycaemic control 
a 
 
a* 
 
Fecal microbiota 
a 
 
a* 
 
Plasma and fecal metabolomics 
a 
 
a 
 
Sleep and physical activity 
a 
 
a* 
a 
Psychosocial  
a 
 
a 
 
Quality of life 
a 
 
a 
a 
Eating behavior and dietary habits   
a 
 
a 
a 
Physical Fitness 
a 
 
a 
 
*Assessment will be conducted during the last 2 weeks of the intervention.  

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3.5. Primary outcome: hepatic fat  
The quantification of hepatic fat will be performed using MRI with a Siemens 3T 
Magnetom Vida scanner located at San Cecilio University Hospital in Granada. 
Additionally, liver steatosis, viscosity and fibrosis severity will be assessed using 
attenuation imaging coefficient, shear wave elastography and shear wave dispersion with 
a Canon Aplio i800 (located at San Cecilio University Hospital in Granada). The Fibrosis-
4 (FIB-4) index will be calculated as an indicator of liver fibrosis severity (22). 
3.6. Secondary outcomes 
Specific fat depots: subcutaneous, visceral and intermuscular adipose tissue, as well as 
pancreatic fat content, will be obtained using MRI. To assess subcutaneous, visceral and 
intermuscular adipose tissue in all 3D abdominal volume (i.e., volume, cross-sectional 
area at selected levels, and mean/median fat fraction), a semiautomatic software program 
will be employed for tissue segmentation. These valuable image markers will be derived 
from a standard 6 echo Dixon series, ensuring accurate characterization of abdominal 
adipose tissue distribution and composition. We will also measure image markers from 
mid-thigh using a 6 echoes Dixon series, and will obtain cross-sectional area, muscular 
tissue, intramuscular adipose tissue, intermuscular adipose tissue, fat fraction, 
subcutaneous adipose tissue, and bone marrow fat fraction. The segmentation for all these 
structures will be performed with a semiautomatic proprietary algorithm. 
Anthropometry and body composition: body weight and height measurements will be 
obtained using a stadiometer and scale (Seca model 799, Electronic Column Scale, 
Hamburg, Germany). Participants will be instructed to be barefoot and wear lightweight 
clothing during these measurements. Neck, waist, and hip circumferences, as well as calf 
girth, will be determined following the procedures outlined by the International Society 
for the Advancement of Kinanthropometry (ISAK) by certified personnel (ID’s ISAK: 
#637686045477240182 and #638227824311214767) (23). Furthermore, fat mass, fat free 
mass, and overall adipose mass will be assessed by bioelectrical bioimpedance (Tanita 
MC 980-MA Plus, Tanita Corporation, Tokyo, Japan), with measurements taken after 
voiding the bladder to ensure accurate readings. Bone mineral density and content will be 
assessed by dual-energy X-ray absorptiometry scans (Discovery Wi, Hologic, Inc., 
Bedford, MA, USA). Participants will be instructed to maintain a fasting period of 8-10 
hours prior to the appointment, during which they will be advised to consume only water 
and abstain from solid food or other beverages. In addition, participants will be advised 
to abstain from alcohol or diuretics 24 hours before the test and to avoid stimulants like 
caffeine for 12 hours before the test, and avoid engaging in moderate exercise or physical 
activity for 24 hours, or vigorous exercise for 48 hours, prior to the test.  
Cardiometabolic risk markers: venous blood samples will be carefully preserved at a 
temperature of -80°C to ensure their integrity for subsequent analysis. We will collect a 
full set of cardiometabolic risk markers, as these parameters may inform about the 
metabolic dysregulation in liver ease (24). These set will include: fasting glucose (Alinity 
C system analyzer, Abbott Laboratories, IL, USA), fasting insulin (UniCel DxI 800 
access immunoassay system, Beckman Coulter Inc., CA, USA), hemoglobin A1c 
(automated glycohemoglobin G11 analyser, Horiba) and lipid profile (i.e., total 
cholesterol, high-density lipoprotein cholesterol, triglycerides, apolipoprotein A1 and B) 
using Alinity C system analyzer (Abbott Laboratories, IL, USA). Low-density lipoprotein 
cholesterol will be calculated using a previously validated equation (LDL-c = CT – HDL-
c – (TG/5) (25).  Furthermore, we will measure alkaline phosphatase (Alinity C system 
analyzer, Abbott Laboratories, IL, USA). Liver markers (i.e., alanine transaminase, 

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aspartate aminotransferase and gamma-glutamyl transferase) will be also measured using 
an Alinity C system analyzer. Inflammatory markers (i.e., C-reactive protein and 
interleukin-6) will be measured using a AU5800 automated analyzer (Beckman Coulter 
Inc., CA, USA). Additionally, we will calculate insulin resistance surrogates, such as 
HOMA-IR. We will also conduct multi-omics (metabolomics, proteomics) analysis on 
plasma samples according to budget considerations. The omics analyses will allow the 
identification of: (i) metabolites/proteins strongly associated to hepatic fat content 
(baseline – cross-sectional analysis); (ii) metabolites/proteins predictors of hepatic fat 
content changes after the intervention. In addition, fasting urine samples will be collected 
for targeted metabolomic analysis to quantify the profile of essential and non-essential 
amino acids, providing complementary data on nitrogen balance and potential indicators 
of metabolic stress or muscle protein catabolism. Finally, systolic and diastolic blood 
pressure will be measured using an automated monitor (M3-Comfort, Omron Healthcare 
Europe B.V., Hoofddorp, The Netherlands), following the established guidelines outlined 
by the 2021 European Society of Hypertension (26). 
Glycaemic control: due to the crucial role of insulin resistance in MASLD development, 
through fatty acid accumulation in hepatocytes, CGM and analysis of acute response to 
glucose will be measured. Participants will be instructed to wear a CGM device 
(FreeStyle LibrePro, Abbott Laboratories, Abbott Park, IL) during 2 weeks before the 
intervention (lead-in period) and during the last 2 weeks of the intervention (weeks 11 
and 12). The CGM data obtained will be used to calculate different variables related to 
glycaemic control (i.e., 24-hour mean glucose), following the guidelines outlined in the 
most recent international consensus statement (27). Additionally, oral glucose tolerance 
tests will also be conducted as part of the glycaemic assessment using a 75-g oral glucose 
dose (NUTER TEC: orange flavour, Toulouse, France). Furthermore, capillary blood 
lactate will be measured using a stationary lactate analyzer (Biosen C-Line, EKF 
Diagnostic GmbH, Germany). 
Fecal microbiota: for the comprehensive identification and quantification of fecal 
microbiome diversity and composition, pre-intervention and during the last 2 weeks of 
the intervention, fecal samples will be collected. Stool microbial DNA will be isolated 
from participants and subsequently, 16S rRNA gene amplicon sequencing, with the 
possibility of employing shotgun methodology pending final budget considerations, will 
be performed. Furthermore, a fecal metabolomic fingerprint analysis will be conducted 
to determine the metabolic profile among different groups of patients (pending final 
budget considerations).  
Sleep and physical activity: sleep quality and chronotype will be evaluated through the 
administration of validated questionnaires, including the Pittsburgh Sleep Quality Index 
(PSQI) (28), Munich Chronotype Questionnaire (MCTQ) (29), and Horne and Östberg 
Questionnaire for Morning-Evening type assessment (MEQ-SA) (30). Objective 
measures of sleep, physical activity levels and rest-activity rhythms will be obtained using 
triaxial accelerometer (ActiGraph GT3X+, Pensacola, FL, USA) that participants will 
wear on their non-dominant wrist during two-week periods: before the beginning of the 
intervention (lead-in period) and during the last two weeks of the intervention (weeks 11 
and 12). Also, steps counts will be assessed by activity band, at baseline and the last 2 weeks 
of intervention.  
Psychosocial assessment: as weight loss intervention may improve emotional well-being 
and psychosocial functioning, several key psychological dimensions will be evaluated 
through the administration of validated questionnaires: the Beck Depression Inventory 

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Fast Screen for depression (BDI-FS) (31), the Perceived Stress Scale for stress (PSS) (32), 
and the State-Trait Anxiety Inventory for anxiety (STAI) (33). 
Quality of life: quality of life may also improve from weight loss intervention (34) and 
will be evaluated using the EuroQol 5 dimensions 5 levels (EQ-5D-5L) questionnaire (35) 
and Rand Short Form 36 (SF-36) (36). In addition, participants will be asked to complete 
an adverse events questionnaire to identify any potential adverse effects or complications 
experienced during the intervention.  
Eating behavior and dietary habits: to assess participants' adherence to the 
Mediterranean dietary pattern, validated questionnaires such as the PREDIMED 
questionnaire will be administered. This questionnaire provides a reliable measure of 
adherence to the specific dietary components and guidelines of the Mediterranean diet 
(37). Food Frequency Questionnaire (FFQ) (38) will be administered to assess the 
frequency that participants have consumed each specific food during the previous 4 
weeks.  
Physical fitness: an incremental treadmill exercise test until exhaustion will be performed 
to determine cardiorespiratory fitness. The modified Balke protocol (41) will be applied, 
which has been extensively used and validated (42–44). The exercise electrocardiogram 
and heart rate (HR) will be monitored continuously and reviewed by a cardiologist. 
Supervised exercise proposed in the present study could restore or mitigate adverse effect 
of diet-induced weight loss on muscle strength (45). Thus, upper muscular strength will 
be assessed through hand grip strength test (46) using a digital hand dynamometer (TKK 
5401 Grip-D; Takei, Tokyo, Japan), whereas lower body muscular strength will be 
assessed through the 30-s sit-to-stand muscle power test (47), and walking speed to assess 
functional capacity with gait speed test (48).  
Confounding: as unintentional reductions in energy intake (10-30% or ~300-500 
kcal/day) have been reported when participants confine their eating windows to 4-10 
h/day (49,50), we will control and analyze changes in energy intake over the intervention 
period. Participants will undergo dietary assessments through the completion of 3 non-
consecutive 24-hour dietary recalls (2 working days and 1 non-working day) (51). These 
recalls will be conducted via face-to-face or telephone interviews by qualified and trained 
research nutritionists. Total energy intake, carbohydrates, fat and protein intake will be 
calculated. In addition, to understand the important sex dimorphism in MASLD 
development, at baseline and after the intervention, specific reproductive-profile 
questions will be asked regarding detailed information on menstrual cycle history and 
hormonal contraceptive use and type, as well as regarding any gynecological condition. 
This will help in categorizing participants into different hormonal profiles, following the 
recent consensus (52–54). 
3.7. Intervention description 
3.7.1. Usual care group 
Participants randomly assigned to the UC group will be indicated to continue with their 
dietary eating time schedule. All participants will receive monthly in-person nutritional 
education sessions, lasting approximately 90 minutes, throughout the intervention for 
weight management and cardiovascular health promotion based on Mediterranean dietary 
patterns (55) and physical activity recommendations from the World Health Organization 
(56). Key points in nutritional counseling will be: a) to correctly interpret food labels and 
to plan grocery, b) to recognize and include high satiety foods, and c) to fight against 
some extended myths in Spanish culture such as “alcohol in small quantities is healthy”, 

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32 
“it is unhealthy to skip breakfast”, or “it is important to eat 5 meals per day”.  
3.7.2. Time-restricted eating intervention 
Before the beginning of the intervention, participants allocated to TRE groups will select 
their preferred 8-hour eating window before the intervention and will be required to 
maintain the same 8-hour eating window during the 12-week intervention. They will be 
advised that the last meal should be completed before or at 21:00. Participants assigned 
to the TRE groups will be strictly prohibited from consuming any calorie-containing food 
or beverage outside their designated 8-hour eating window. However, they will be 
allowed to consume water, coffee, and tea without sugar or artificial sweeteners during 
the fasting period. Instructions for the TRE intervention will emphasize its daily 
implementation, meaning participants are expected to adhere to the eating window 
restriction (±30 minutes) throughout all seven days of the week. 
3.7.3. Supervised exercise intervention 
The objective of the supervised exercise program will be to ensure its transferability and 
feasibility for the target population. We will follow the Consensus on Exercise Reporting 
Template to facilitate replicability and transparency (57) and record the timing when 
participants are training (58). Thus, the physical activity recommendations for adults 
proposed by the World Health Organization serve as the foundation for determining the 
specific exercise dosage in TEMPUS (59). Given that both resistance and aerobic training 
modalities have shown improvements in hepatic steatosis among patients with MASLD 
(60,61), the TEMPUS study will combine both supervised resistance and aerobic high-
intensity interval training (HIIT) sessions. Qualified sports scientist from outside the 
research group will carefully supervise the exercise sessions and work with groups of no 
more than six people to ensure that participants perform the exercise technique correctly, 
and at proper intensity. Moreover, participants will receive an individualized moderate-
intensity goal-setting aerobic (i.e., walking) program consisting of increasing 10% daily 
steps per week based on their daily steps logs. Goal-setting aerobic will be updated 
weekly using a smart band (Xiaomi Mi Band 7, Xiaomi, Pekin, China) that participants 
enrolled in the exercise groups will wear on their wrists during the intervention period.  
Volume: participants will engage in a 12-week intervention program including 2 
supervised exercise sessions per week ranging from 60 to 90 minutes per session. 
Furthermore, participants will receive instructions to complete their personalized daily 
step goals established for each day.  
Intensity: supervised sessions will start with a supervised circuit-based resistance training 
that focuses on upper and lower body exercises that target major muscle groups. To 
prescribe intensity, the rate of perceived exertion (RPE) scale ranging from 0 to 10 will 
be utilized, using the evaluations and first sessions to properly train the participants on 
this scale (62). The target intensity for resistance training will be set at an RPE level 
greater than 7, which will vary depending on the week number of the intervention (from 
7 to 10). The participants will be encouraged to achieve this intensity through all the 
exercises performed in each lap of the circuit. Moreover, training sessions will include 
HIIT as the vigorous-intensity aerobic exercise component. HIIT will consist of 3-4 sets 
of 4-minute intervals at >85% of the individual heart rate reserve (HRR) with 4 minutes 
of active recovery at 65-75% HRR. HRR will be calculated considering the peak of HR 
achieved in the incremental treadmill test and the rest HR lying in bed recorded during 
pre-intervention assessments. HR will be continuously monitored during all exercise 
sessions using the Polar H10 band (Polar Products Inc., Stow, OH, USA). 

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33 
Frequency: participants will perform 2 supervised exercise training sessions per week, 
with a resting period of at least 48 hours between them. Additionally, participants will be 
instructed to accomplish the prescribed daily step goal. If a participant misses a training 
session, it will be rescheduled and recovered considering a minimum resting period of 48 
hours between sessions. 
Type of exercise: the resistance training sessions will be composed of major upper and 
lower body muscle groups including (63): horizontal/vertical pull exercises (i.e., seated 
low row and lat pulldown with resistance elastic bands), hip-dominant exercises (i.e., 
deadlift with resistance elastic bands and weight-bearing glute bridge), knee-dominant 
exercises (i.e., weight-bearing squats and lunges), and horizontal/vertical push exercises 
(i.e., weight-bearing push-ups and shoulder press with resistance elastic bands). We will 
propose 4 levels of exercises’ difficulty based on the resistance elastic band and the 
progressive complexity of the basic movement patterns involved. After the resistance 
circuit training, HIIT will be preferably performed on a treadmill to ensure a suitable 
progressive overload. As alternative modality, elliptical ergometer will be used. 
Training load variation: we are aware that participants might not be immediately capable 
of exercising at high intensities and volumes. Therefore, there will be a gradual 
progression to the assigned exercise dose in 3 phases (see Figure 2) and proper technique 
will be a priority for the difficulty level progression to avoid potential injuries. 
Training periodization: the supervised exercise intervention will be divided into 3 phases 
(Figure 2). The initial phase will have a length of 6 weeks and will start with a first week 
of familiarization. During this week, participants will learn the structure and organization 
of the sessions and the movement patterns that constitute the base of the different 
exercises. Considering that an inactive person may be unable to immediately train at the 
selected doses, the familiarization phase will prepare participants to gradually increase 
the workload until the required dose is achieved. On the other hand, the resistance training 
will set light loads using resistance elastic bands or weight-bearing exercises. They will 
increase the load and coordinative difficulty (through 4 levels for each exercise) as soon 
as participants perform the exercises with a proper technique.  
We anticipate that both resistance training and HIIT load will be parallelly higher with 
the participants fitness increase. In this context, HIIT will be controlled based on HRR. 
Higher speed or power will need to be selected to achieve a determined percentage of 
HRR when fitness levels are increasing. 
Training session: a training session will be organized as follow: (i) a warm-up of one 
circuit training lap with light loads using resistance elastic bands or weight-bearing 
exercises (20 seconds work: 20 seconds rest), (ii) 2 to 4 circuit training laps of eight 
exercises (30 seconds work: 30 seconds rest) at 7-10 RPE, with a between-set rest of 3-5 
min, and (iii) 3 or 4 sets of  4-min intervals at 85-95% HRR interposed by 4 min of active 
recovery at 65-75% of HRR, with 2 min prior of warm-up. A cool-down protocol of 5 
min will be performed at the end including anterior and/or posterior chain exercises for 
muscle elongation and/or relaxation. The total duration of exercise sessions will be 55 
minutes in the familiarization week, 65 minutes during the first phase, 75 minutes in the 
second phase, and 85 minutes in the third phase.  

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34 
 
Figure 2. Supervised exercise periodization of the TEMPUS project. *Week of familiarization. HIIT, high-
intensity interval training; HRR, heart rate reserve; RPE, rate of perceived exertion; RT, resistance training. 
AET, aerobic endurance training. 
3.8. Participant retention, adherence and sustainability 
The Principal Investigator and study team will exert every effort to facilitate participants' 
completion of all study visits and ensure overall study retention. The following strategies 
will be implemented to maximize participants' retention and minimize loss to follow-up: 
a) implementing a proactive retention plan that focuses on building close participant 
relations and ensuring participant satisfaction, b) giving the opportunities for participants 
and their families to ask questions and voice any concerns related to their condition 
throughout the study, c) reinforcing comprehension of the objectives and protocol of the 
study during study visits or conducting question and answer sessions after each visit, and 
d) evaluating each likelihood of dropout and implementing appropriate interventions to 
maintain their interest and motivation to continue participating in the study. 
All supervised exercise sessions will be performed in a well-lit and airy room, providing 
to the participants the opportunity to choose their own music. The training specialists and 
other study staff will consistently offer support to participants throughout the duration of 
the study. 
During the 12-week intervention period, participants will be required to record their daily 
sleep and eating times (i.e., exact times of the beginning of the first meal and of the end 
of the last meal), as well as any potential adverse events in a mobile phone app specifically 
designed for the study. These data will be revised 2 to 3 times every week, asking the 
participant for missing records, and will provide insights into their adherence to the 
prescribed eating window. For participants in the TRE + Exercise group and TRE alone 
group, each day will be labeled as adherent if the participant meets their self-selected 8-
hour (±30 minutes) eating window. Adherence will then be defined as the percentage of 
days on which the eating window was met. Finally, we will assess the long-term 
adherence to the intervention (at the 12-month follow-up). This will allow us to evaluate 
the sustainability of participants' adherence over an extended period. Attendance will be 
defined as percentage of exercise sessions attended by the participant (TRE + Exercise 
 
Phase 
FIRST 
SECOND 
THIRD 
Week 
1* 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
Supervised Exercise Volume (min/week) 
110 
130 
130 
130 
130 
130 
150 
150 
150 
150 
170 
170 
Warm-up (RT) 
Volume (min/session) 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
Resistance training 
Total volume (min/week) 
32 
32 
32 
32 
32 
32 
48 
48 
48 
48 
64 
64 
Intensity  
(RPE 1-10 scale) 
7 
7 - 8 
7 - 8 
7 - 8 
8 - 9 
8 - 9 
8 - 9 
8 - 9 
9 - 10 
9 - 10 
9 - 10 
9 - 10 
Secs work/Secs rest 
(per exercise) 
30/30 
30/30 
30/30 
30/30 
30/30 
30/30 
30/30 
30/30 
30/30 
30/30 
30/30 
30/30 
Number of laps 
(per session) 
2 
2 
2 
2 
2 
2 
3 
3 
3 
3 
4 
4 
Rest between laps (min) 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
Number of exercises  
(per session) 
8 
8 
8 
8 
8 
8 
8 
8 
8 
8 
8 
8 
Rest between 
RT & AET 
Volume (min/session) 
3 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
Warm-up (HIIT) 
Volume (min/session) 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
HIIT 
Vigorous-intensity 
intervals / Moderate-
intensity intervals 
3/3 
4/4 
4/4 
4/4 
4/4 
4/4 
4/4 
4/4 
4/4 
4/4 
4/4 
4/4 
Total volume vigorous-
intensity (min/week) 
24 
32 
32 
32 
32 
32 
32 
32 
32 
32 
32 
32 
Intensity (%HRR) 
80 - 85 
85 - 95 
85 - 95 
85 - 95 
85 - 95 
85 - 95 
85 - 95 
85 - 95 
85 - 95 
85 - 95 
85 - 95 
85 - 95 
Total volume moderate-
intensity (min/week) 
24 
32 
32 
32 
32 
32 
32 
32 
32 
32 
32 
32 
Intensity (%HRR) 
60 - 65 
65 - 75 
65 - 75 
65 - 75 
65 - 75 
65 - 75 
65 - 75 
65 - 75 
65 - 75 
65 - 75 
65 - 75 
65 - 75 
Cool down 
Volume (min/session) 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 

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and Exercise alone groups), recorded by the trainers, divided by the actual number of 
exercise sessions offered (12 weeks x 2 sessions / week = 24). Finally, we will assess the 
long-term adherence to the intervention (at the 12-month follow-up). This will allow us 
to evaluate the sustainability of participants' adherence over an extended period.  
3.9.  Adverse events 
The study coordinators and project managers will oversee the collection of data and 
monitor the frequency of reported adverse events on a weekly basis using the mobile 
phone app (Tempus: com.nnbi.app_extreme_granada; NNBi2020 S.L., Navarra, Spain). 
Additionally, participants will complete validated questionnaires that assess 
gastrointestinal and autonomic symptoms, well-being, eating behavior, sleep quality, 
stress levels, mood, anxiety, and depression. These questionnaires will offer valuable 
information on any potential adverse effects and overall health-related outcomes. If a 
serious adverse event or an unanticipated problem occurs, the study coordinators will 
immediately notify both the principal investigators and the medical staff. Subsequently, 
a collective decision will be made and, if needed, the ethics committee will be properly 
informed. Moreover, appropriate measures will be taken to address and manage the 
reported event effectively. 
3.10. Data management 
Data collected will be directly entered into REDCap (Research Electronic Data Capture), 
a secure web-based platform specifically designed to create and manage research-related 
databases and surveys. This platform will ensure data security and confidentiality. For 
any data not recorded in REDCap, strict access control measures will be implemented to 
securely store the data on university computers, maintaining confidentiality and data 
integrity. 
To ensure data quality and integrity, regular quality control checks will be conducted to 
identify any potential data anomalies, such as missing data or forms, data that falls outside 
the expected range, erroneous data entries, illogical dates over time, data inconsistencies 
across different forms and study visits, and incomplete fields on completed forms without 
a valid explanation for the missing data. Any identified issues will be promptly reviewed 
and addressed by the data monitoring committee to ensure the accuracy and reliability of 
the data. The data monitoring committee will be independent from the sponsor. There are 
no auditing procedures planned.  
3.11.  Patient and public involvement  
TEMPUS will stimulate patient and public involvement throughout the entire process. 
Potential participants have actually been involved in the preparation of the intervention 
and in the development of the mobile app, organization of the outcome measures, as well 
as are helping in the recruitment of new participants. Once we have the results ready, we 
will actively involve the participants in the reporting and advocacy of the study results. 
4. Statistical Analysis Plan 
4.1. Randomization and blinding 
We expect to recruit and randomize ~184 participants using both stratification and 
permuted blocks with random block sizes, after completing the pre-intervention 
measurements. Randomization will be stratified based on sex (men-women), resulting in 
2 strata. Prior to the beginning of the trial, randomization lists will be generated for each 
strata. The block sizes will be randomly determined, with allowable sizes of 4 and 8. 

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Within each block, random selection will be used to assign participants to one of the four 
possible groups (i.e., UC, TRE, Exercise or TRE + Exercise) using a parallel design with 
a 1:1:1:1 allocation ratio. The sequential assignment of participants will follow the 
predetermined randomization list specific to their strata. The utilization of random block 
sizes ensures that the next assignment cannot be predicted. Stratification by sex ensures 
that the intervention groups will be balanced for this important characteristic. Blinding 
procedures will be rigorously applied to all personnel responsible for assessing primary 
outcomes, specifically hepatic fat depots and other ectopic fat depots derived from MRI. 
This blinding will also extend to those analyzing cardiometabolic risk factors, conducting 
glucose monitoring, examining fecal microbiota, evaluating physical activity and 
sedentary time, as well as those engaged in the statistical analysis of data. Conversely, 
personnel responsible for other measurements and intervention administration will not be 
blinded to the group assignment (open-label). Participants will receive clear information 
about the group they will be assigned to, along with details about the study hypotheses. 
In order to ensure practicality and feasibility, and based on previous research experience 
(64–66), the study will be conducted in successive waves of participants, each of them 
including a maximum of 16 participants. 
4.2. Sample size 
Based on previous findings from a recent trial on the combination of alternate day fasting 
and exercise on hepatic fat content (67), we anticipate approximately 5.0% reduction in 
hepatic fat content in the TRE + Exercise group, 2.5% in the TRE group, 2.5% in the 
exercise group, and no significant change in this outcome in the usual-care group. 
Assuming a pre-post correlation of 0.8 and a standard deviation of 6 points in the main 
outcome, we estimate a medium effect size of 0.45. To detect this effect size as 
statistically significant in a one-way ANOVA with α = 0.05 and a power of 0.8, a 
minimum of 19 patients per group is required. Accounting for subgroup analyses based 
on sex and a maximum dropout rate of 20%, we will aim to recruit ~46 participants for 
each trial group, resulting in a total sample size of ~184 participants, with ~92 women 
included. To ensure a balanced representation of both sexes and maintain an adequate 
sample size, we will implement several strategies: 
- 
Recruitment Process: A specific recruitment process will be utilized to aim for an 
equal enrollment of men and women. For each woman recruited, we will 
encourage her to invite one man to participate in the study. This approach has been 
successfully employed in our previous intervention studies and has proven 
effective in achieving a balanced sex distribution (68–70). 
- 
Sample size calculations: Our sample size calculations have taken into account 
subgroup analyses by sex. We have conservatively estimated a maximum dropout 
rate of approximately 20%. By considering this dropout rate, we have ensured that 
our study is adequately powered to detect the specified effect size even if there 
are differential dropout rates between men and women. For example, if men have 
a dropout rate of 5% and women have a dropout rate of 15%, our study will still 
have sufficient power to analyze the data separately in men and women. 
- 
Expected dropout rate: While we have conservatively estimated the maximum 
dropout rate, we anticipate that the actual dropout rate will be relatively lower and 
similar between both sexes. This expectation is based on our previous studies and 
the measures we have to promote participant engagement and adherence (68–70). 
4.3. Statistical analysis 
The main analyses will consist of the intention-to-treat analyses for the primary and 

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secondary outcomes using a constrained baseline (meaning baseline adjusted) linear 
mixed model, which accounts for baseline differences among the study groups. The 
model will include fixed effects for time (three levels) and treatment (four levels) as well 
as the unique participant identifier as a random effect, and sex will be included as a 
covariate. Model assumptions will be checked. In case we detect model violations, we 
will take appropriated measures by e.g., conducting data transformations. If the global 
test of significance indicates between-group differences, pairwise comparisons will be 
explored. Although no adjustments for multiplicity will be performed, family-wise type 
1 error rate on the primary outcome will be retained by using a hierarchical analytic 
approach. The prespecified hierarchical hypotheses will be tested using the prespecified 
sequence: TRE + Exercise versus UC, TRE versus UC, Exercise versus UC, TRE + 
Exercise versus TRE, TRE + Exercise versus Exercise and TRE versus Exercise. The 
main statistical analyses will be performed by an independent researcher (Dr. Almudena 
Carneiro-Barrera), who is not involved in the recruitment, evaluations, and interventions, 
and will be performed blinded to the treatment allocation by coding the intervention arms 
(e.g., A, B, C, D). 
It should also be noted that the intervention effect assessments will not only be based 
on statistical and practical significance (as usually done), but also on a practical benefit 
approach emphasizing and reporting unadjusted values that are intuitive to human 
judgment and readily replicable considering the design and methodology of this project. 
 
5. Ethics and dissemination 
The study has received ethical approval from the Granada Provincial Research Ethics 
Committee (CEI Granada - 0365-N-23) and will be performed following the ethical 
guidelines of the Declaration of Helsinki. Before their inclusion in the study, participants 
will be required to provide oral and written informed consent, with further details 
available in the Online Supporting Information. 
Results will be presented in peer-reviewed, scientific journals and at international 
conferences. We aim to publish a main paper with the primary outcome data. Since it is 
a large study with numerous secondary outcomes, other specific manuscripts on each 
topic will be published. 
 
6. References 
1. 
WHO Regional office for Europe. WHO European Regional Obesity Report 2022. 2022. 
1–220 p.  
2. 
Tan CY, Vidal-Puig A. Adipose tissue expandability: The metabolic problems of obesity 
may arise from the inability to become more obese. Biochem Soc Trans. 2008;36(5):935–
40.  
3. 
Mantovani A, Scorletti E, Mosca A, Alisi A, Byrne CD, Targher G. Complications, 
morbidity 
and 
mortality 
of 
nonalcoholic 
fatty 
liver 
disease. 
Metabolism. 
2020;111:154170.  
4. 
Rinella ME, Lazarus J V., Ratziu V, Francque SM, Sanyal AJ, Kanwal F, et al. A multi-
society Delphi consensus statement on new fatty liver disease nomenclature. J Hepatol 
[Internet]. 2023 Jun [cited 2023 Jun 26];0(0). Available from: http://www.journal-of-
hepatology.eu/article/S016882782300418X/fulltext 
5. 
Armstrong MJ, Adams LA, Canbay A, Syn WK. Extrahepatic complications of 
nonalcoholic fatty liver disease. Hepatology [Internet]. 2014 Mar 1 [cited 2023 Jul 
21];59(3):1174–97. 
Available 
from: 

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https://onlinelibrary.wiley.com/doi/full/10.1002/hep.26717 
6. 
Ganguli S, DeLeeuw P, Satapathy SK. <p>A Review Of Current And Upcoming 
Treatment Modalities In Non-Alcoholic Fatty Liver Disease And Non-Alcoholic 
Steatohepatitis</p>. Hepat Med [Internet]. 2019 Nov 15 [cited 2023 Jun 14];11:159–78. 
Available 
from: 
https://www.dovepress.com/a-review-of-current-and-upcoming-
treatment-modalities-in-non-alcoholic-peer-reviewed-fulltext-article-HMER 
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7. Summary of changes to Study Protocol 
 
Date 
Amendment 
01/07/2024 
Review and improvement of the 
statistical analysis methods, including 
constrained baseline (meaning baseline-
adjusted) linear mixed model. 
01/07/2024 
Update of secondary outcomes including 
new outcomes derived from magnetic 
resonance imaging, elastography, fasting 
urine, and fasting blood samples.