Article Text
PDF
Abstract
Introduction Dyslipidaemia, affecting approximately 39% of adults worldwide, is a major risk factor for cardiovascular disease. Individuals with dyslipidaemia are often prescribed statins, which effectively lower plasma low-density lipoprotein cholesterol (LDL-C), thereby reducing the risk of cardiovascular events and mortality. Although statins lower LDL-C, emerging evidence suggests that they may counteract the beneficial adaptations to exercise in skeletal muscle mitochondria and whole-body aerobic capacity. The underlying mechanisms remain unclear, and there is a need for studies investigating how statins influence molecular adaptations to exercise. The primary objective of this study is to investigate the combined effects of statin therapy and focused exercise training on mitochondrial function and whole-body aerobic capacity in people with dyslipidaemia. The untargeted proteomic analysis will be incorporated to provide detailed insights into how statins may affect mitochondrial proteins and other muscle metabolic traits, offering molecular explanations for altered functional readouts at both the muscle and whole-body levels.
Methods and analysis A total of 100 women and men (aged 40–65 years) diagnosed with dyslipidaemia without atherosclerotic cardiovascular disease will be enrolled in this 12-week, double-blinded, randomised, placebo-controlled trial. Participants will be randomised into one of four groups using a block randomisation approach to ensure an allocation ratio of 60:40 for exercise and non-exercise conditions, respectively. The four groups will be: (1) exercise+placebo, (2) exercise+atorvastatin (80 mg/day), (3) atorvastatin (80 mg/day) and (4) placebo. The primary outcome is mitochondrial function, measured by changes in skeletal muscle citrate synthase activity from baseline to post-intervention. Secondary outcomes include whole-body aerobic capacity (VO2peak) and proteomic analyses. Genetic analysis will be conducted to assess the role of genetic polymorphisms in individual responses to statins and exercise.
Ethics and dissemination The trial has received ethical approval from the Faroe Islands Ethical Committee (2024-10) and adheres to the Declaration of Helsinki and General Data Protection Regulation (GDPR). Results will be published in peer-reviewed international journals.
Trial registration number NCT06841536.
- Exercise
- GENETICS
- Physiology
- Hydroxymethylglutaryl-CoA Reductase Inhibitors
- Randomized Controlled Trial
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.
Statistics from Altmetric.com
STRENGTHS AND LIMITATIONS OF THIS STUDY
Four-arm, randomised, double-blinded, placebo-controlled design enabling isolated and combined assessment of high-dose statin and supervised exercise effects.
Block randomisation stratified by sex and prior statin use ensures balanced allocation across intervention arms.
Comprehensive skeletal muscle phenotyping using untargeted proteomic analysis of biopsies captures molecular adaptations to interventions.
Integrated genetic analysis of statin-metabolism polymorphisms informs individual variability in drug and exercise responses.
Exclusion of patients with atherosclerotic cardiovascular disease and a 12-week intervention period may limit generalisability and capture of longer-term adaptations.
Introduction
Dyslipidaemia, a medical condition affecting approximately 39% of adults aged 25 years or older globally,1 is characterised by an imbalance in lipid levels, including elevated serum concentrations of total cholesterol, low-density lipoprotein cholesterol (LDL-C) and triglycerides, or decreased levels of high-density lipoprotein cholesterol.2 Elevated LDL-C levels alone contributed to an estimated 4.4 million deaths and 98.6 million disability-adjusted life years in 2019,1 underscoring the significant public health burden posed by dyslipidaemia worldwide.
Regional variability in prevalence is notable, with Europe reporting the highest rates of raised plasma cholesterol levels (54% for both sexes).1 LDL-C plays a key role in transporting cholesterol to the artery walls, where its accumulation leads to the formation of atherosclerotic plaques.3–6 Persistent elevation of LDL-C is a hallmark of dyslipidaemia and is strongly associated with an increased risk of premature atherosclerotic cardiovascular disease (ASCVD).5 6 Therefore, reducing systemic LDL-C levels remains a primary therapeutic goal in preventing and treating ASCVD.3
Statins, or hydroxymethylglutaryl-coenzyme A reductase inhibitors, are commonly prescribed to individuals with dyslipidaemia. They effectively reduce LDL-C levels and significantly lower the risk of cardiovascular events.7 A 1 mmol/L reduction in LDL-C achieved through statin therapy is linked to a 10%–20% decrease in cardiovascular events and all-cause mortality.8 9 Exercise training is another recommended intervention, as it also lowers LDL-C levels and offers additional cardiovascular benefits, thereby reducing the risk of disease and mortality.10 11 Even modest improvements in cardiorespiratory fitness (CRF) of ~1 MET, achievable with endurance training in most individuals, are associated with a 10%–25% improvement in survival rates.12 Given the rising prevalence of cardiovascular diseases worldwide, understanding how common treatments like statins interact with lifestyle interventions such as exercise is critical for optimising patient outcomes.
While both statins and exercise offer distinct cardiovascular benefits, emerging evidence suggests that their combination may not always yield the expected synergistic or additive effects. Statin therapy may blunt some of the beneficial effects of exercise training, particularly regarding improvements in cardiorespiratory capacity and skeletal muscle mitochondrial function.13 For example, simvastatin (40 mg/day) diminished the increase in skeletal muscle citrate synthase (CS) activity and whole-body aerobic capacity accrued with 12 weeks of endurance exercise in participants with overweight.13 Conflicting evidence exists,14 but similar findings have been reported by Ryan et al,15 who demonstrated that high-dose atorvastatin (80 mg/day) alone progressively reduced skeletal muscle mitochondrial oxidative capacity in overweight but otherwise healthy individuals. These results align with mounting evidence that statins can negatively affect skeletal muscle mitochondria.13 16–28 The exact mechanisms by which statins interfere with skeletal muscle mitochondria and exercise-induced adaptations remain unclear, but proteomic analysis may provide new insights into the underlying pathways.
In addition, statins are associated with a dose-dependent rise in new-onset type 2 diabetes, particularly among high-intensity statin users and individuals already at increased risk.29–31 The potential mechanisms include reductions in insulin sensitivity and impairments in beta-cell function.32 33 Notably, recent evidence suggests that these metabolic effects of statins may differ between men and women.33 Collectively, these processes underscore the complex metabolic impact of statins, particularly at higher doses. While statins provide significant cardiovascular benefits, these findings underscore the need for careful patient selection and glucose monitoring, especially for those already at high diabetes risk.34
Despite being generally safe, statins are associated with muscle-related side effects, known as statin-associated muscle symptoms (SAMS). SAMS, which affect 5%–30% of statin users, include muscle pain, muscle weakness and cramps.35–37 These symptoms can lead to discontinuation of therapy, which negatively impacts ASCVD outcomes. SAMS may also reduce the quality of life for patients and discourage physical activity.38 Several studies indicate that physical activity exacerbates SAMS, leading to a more sedentary lifestyle in statin users.16 39–42 While the mechanisms behind SAMS remain elusive, some evidence points to statin-induced inhibition of mitochondrial complexes III and IV, as well as coenzyme Q10 deficiency.15 18 43
Additionally, genetic polymorphisms can affect how statins are metabolised, influencing their pharmacodynamics and pharmacokinetics, which in turn impacts muscle exposure to statins.44 However, the extent to which these genetic differences affect the interaction between statin therapy and exercise-induced adaptations in muscle mitochondria and whole-body aerobic capacity is yet to be explored.
In summary, while statins are effective in lowering LDL-C and reducing cardiovascular risk, they may also diminish the beneficial effects of exercise on muscle mitochondria and CRF. Statins may even reduce adherence to physical activity due to SAMS, further complicating the management of dyslipidaemia. Additionally, growing evidence highlights a potential link between statin therapy and the onset of type 2 diabetes, particularly in high-risk populations, warranting further exploration of their metabolic impacts. Despite extensive research, the precise mechanisms behind these effects remain unknown. Importantly, no randomised, double-blinded, placebo-controlled studies have investigated the combined effects of statins and exercise on cardiovascular, muscular and blood-based health markers in individuals with dyslipidaemia, incorporating deep phenotyping and genetic analysis.
Objective
The primary objective of this study is to investigate how statin therapy and exercise training, individually and in combination, affect cardiorespiratory capacity and markers of mitochondrial function, in individuals with dyslipidaemia without ASCVD. Through proteomic analysis, the study aims to provide molecular insights into how statins may alter exercise-induced adaptations in mitochondrial proteins and muscle metabolic traits. A subanalysis will be performed to further explore whether genetic polymorphisms related to the pharmacodynamics and pharmacokinetics of statins influence the interaction between statin therapy and exercise-induced adaptations at both the muscle and whole-body levels.
Methods and analysis
Participants, interventions and endpoints
This study protocol describes a double-blinded, randomised, placebo-controlled, longitudinal trial. The study design is outlined in figure 1.
Schematic overview of the study design. HIIT, high-intensity interval training.
Study setting
All clinical investigations will be conducted at the National Hospital of the Faroe Islands and University of Faroe Islands.
Expected timeline
Recruitment of participants is planned to be initiated in May 2025, and the last participant’s last visit is expected to be in December 2025.
Participant and recruitment
We expect to enrol 100 participants (50 men and 50 women), aged 40–65 years, registered with a plasma LDL-C >4.0 mmol/L measured between November 2018 and April 2024 and without a history of ASCVD as defined by Borg et al.45 46
Recruitment of participants from the Faroe Islands will be conducted in collaboration with medical doctors who have access to the Faroese patient register. Individuals who are interested in participating in the study will receive a thorough introduction to the study and a letter inviting them to attend a medical screening to assess eligibility (see box 1) before inclusion in the study. Informed consent or assent from potential participants will be obtained in person by TS and HE (see online supplemental file 1 for the example consent form). The medical screening consists of a comprehensive evaluation of the individual’s medical history and lifestyle. The letter of invitation will contain general information about the study, including its aims, methods, risk assessments and ethical considerations. Individuals not responding to the invitation letter within 2 weeks will be phoned a maximum of three times.
Supplemental material
Eligibility criteria for study participation
Inclusion criteria
Age: 40–65 years.
LDL-C >4.0 mmol/L, calculated via the Friedewald equation (LDL-C=Total Cholesterol−(HDL-C+0.45×Triglycerides)).
Exclusion criteria
Diagnosed with serious chronic disease including type 1 or 2 diabetes.
Cancer.
A history of atherosclerotic cardiovascular disease.
A history of major depression or other severe psychiatric disorders.
Severe renal dysfunction (creatinine clearance <30 mL/min).
Severe hepatic impairment, defined as alanine-aminotransferase ≥3×the upper limit of normal.78
Active pregnancy or breastfeeding.
Active cigarette or e-cigarette smoker.
Regular (>2 hours per week) aerobic high-intensity exercise training.
HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol.
Individuals with familial hypercholesterolaemia (FH) who meet all other inclusion and exclusion criteria will be enrolled. We will document FH status at screening and apply the same 4-week statin wash-out and randomisation procedures to all participants. If participants with FH comprise a sufficient subgroup, we will perform exploratory analyses to determine whether FH modifies the effects of statin therapy, exercise or their combination. Throughout the trial, the on-study physician will monitor lipid levels and clinical status and may pause or discontinue participation on a case-by-case basis if safety concerns arise.
Participant involvement
No members of the public or participants have been involved in the research question, the design or the conduct of the research. On completion of the data analysis, all participants will receive a lay summary of the study’s main findings.
Participant withdrawal
Participants are free to withdraw from the intervention at any time without reprisal. The study team may also decide to discontinue a participant’s involvement in the study due to safety concerns. All participants, regardless of adherence or withdrawal from the intervention, will be encouraged to complete the end-of-trial assessments.
Allocations
On successful inclusion in the study, participants will be randomly assigned in a 60:40 allocation ratio to either an exercise or non-exercise condition, that is, 12 weeks with (1) exercise+placebo (n=30), (2) exercise+atorvastatin (n=30), (3) atorvastatin only (n=20) and (4) placebo (n=20). An independent statistician will perform a computer-generated randomisation schedule in the 60:40 ratio, stratified by two factors: (1) sex (male or female) and (2) whether participants were on active statin treatment prior to enrolment (yes or no). Although there are no specific inclusion or exclusion criteria regarding prior statin type or dose, this stratification accounts for potential lingering effects that may not fully normalise within the 4-week washout period and for possible differences in side-effect profiles (eg, habituation) between previously treated and statin-naive participants. Following the baseline measurements, a non-blinded colleague with no association with the study will assign the allocation to each study participant in person.
Rationale for the 60:40 ratio
A larger proportion of participants are assigned to the exercise arms to anticipate higher dropout rates from the demanding high-intensity training, thereby preserving adequate power for our key between-exercise-group comparisons (exercise+placebo vs exercise+atorvastatin).
Blinding
Participants will be blinded with regards to the study medication but not exercise training, whereas investigators and outcome assessors will be blinded to both medical treatment and exercise/non-exercise. The blinding will be kept until the completion of the final clinical investigation. For participants’ safety, the physician responsible for medical supervision of the trial is non-blinded to study medication and exercise/non-exercise, but he will not be involved in any clinical investigations or statistical analysis of study outcomes. The effectiveness of participant blinding in each trial group will be assessed by the Bang blinding index (BI),47 which will be calculated based on questionnaire results completed post-intervention. The Bang BI ranges from −1 to 1, with −1 signifying that all study participants guessed the incorrect treatment, 0 signifying that all participants guessed randomly and 1 signifying that all study participants guessed the correct treatment. Successful blinding to study medication is achieved if the 95% CI includes the null value.
Placebo composition
The placebo tablet contains calcium carbonate and the same inactive excipients as the atorvastatin tablet. It is manufactured to be identical in size, shape, colour and packaging to ensure blinded administration for participants and investigators.
Unblinding
Unblinding will be allowed under special circumstances such as addressing a participant’s adverse event; treatment of a participant during a medical emergency that necessitates awareness of their treatment assignment; handling a supposed unexpected serious adverse event and if a household member, such as a child, accidentally ingests the participant’s study medication.
Wash-out period
During the 4-week wash-out period prior to baseline testing, participants will be instructed to discontinue statins treatment and/or any other form of cholesterol-lowering treatment. This is to ensure that potential adverse effects of statins on mitochondrial content and function are normalised prior to the intervention and to secure a stable baseline across trial groups. However, it should be noted that the timescale for normalising mitochondrial content and function following cessation of statin therapy is not clearly defined.
Interventions
Statins or placebo
Atorvastatin (80 mg) or placebo will be ingested once daily as an oral tablet. The initial dosage is set at 40 mg per day for the first week with the maintenance dosage of 80 mg per day starting from the second week. The titration protocol may be extended for participants experiencing intolerable side effects. Under special circumstances, participants who experience intolerable side effects at 80 mg may remain on a lower dosage (40 mg), although we intend to reach 80 mg for all enrolled participants.
Exercise training
Participants will undergo a 12-week, supervised aerobic interval training programme on cycling ergometers, lasting ~40 min per session, 3–4 times weekly. This time-constrained approach emphasises short, high-intensity intervals—an evidence-based strategy for enhancing both muscle oxidative capacity (primary outcome) and cardiovascular capacity (secondary outcome).48 49 Notably, high-intensity interval training is often perceived as more enjoyable than moderate-intensity continuous exercise, potentially enhancing adherence.50 To reduce perceived exertion, we employ passive recoveries between work intervals,51 52 alongside guidance on fueling, hydration and cooling.
At baseline, each participant will perform an incremental exercise test to exhaustion to determine maximal incremental exercise power output (Wmax), which is used to tailor training intensity. Additionally, a critical power profile is established during the first training session via a 5-min time trial and reassessed in weeks 3, 6 and 9 to ensure progressive overload.
Training is structured into four consecutive 3-week blocks, beginning with three sessions per week for the first 6 weeks, then progressing to four sessions per week from week 7 to 12. Alongside the increase in session frequency, the intensity distribution is also ramped up throughout the intervention (see figure 2). This approach allows participants to gradually adapt to both the volume and intensity of training. Each session includes a 5–10 min warm-up, 20–30 min of intervals and a 5–10 min cool-down. Intensity zones range from light (≤45% Wmax; rate of perceived exertion (RPE ~2–3) to moderate (46–63% Wmax; RPE ~4–5) to vigorous (≥64% Wmax; RPE ≥7), with sprint intervals (30–180 s work bouts at >90% Wmax, 15–180 s recoveries) integrated to maximise time above 90% Wmax.48 53 54 We target a weekly progression of >5% in accumulated time spent above Wmax, while simultaneously evaluating each participant’s training load via heart rate (Polar Teampro, Polar Electro Oy, Kempele, Finland), RPE (modified Borg scale) and self-reported well-being (5 point visual analogue scale). Participants allocated to the non-exercise arms (placebo-only and atorvastatin-only) will maintain habitual activity levels.
Overview of the 12-week aerobic interval training programme, illustrating the progressive increase in session frequency (3–4 sessions/week) and the distribution of training intensity (light, moderate and vigorous) across four consecutive 3-week blocks. This design ensures gradual progress.
Endpoints
Primary endpoint
The primary endpoint is the change in mitochondrial content, determined as skeletal muscle CS maximal activity, from baseline (V1) to the end of the intervention (V2).
Secondary endpoints
The secondary endpoints are changes in peak oxygen uptake (VO2peak), markers of metabolic health (lipids, resting blood pressure, glycated haemoglobin A1c, fasting blood glucose, insulin sensitivity and β-cell function, waist circumference and body weight), body composition, hand-grip strength, quality of life, statin accumulation in muscle tissue, SAMS, steady-state systemic oxygen uptake, markers of skeletal muscle oxidative and glycolytic capacity, markers of skeletal muscle mitochondrial biogenesis and skeletal muscle coenzyme Q10 from baseline (V1) to end of intervention (V2). Finally, skeletal muscle proteomics targeting towards outcomes of interest related to oxidative/glycolytic pathway expression are also key secondary endpoints that will provide detailed insights into how statins affect exercise-induced changes in mitochondrial proteins as well as other muscle metabolic traits to provide molecular explanations for potentially altered functional readouts at both the muscle and whole-body levels.
Exploratory endpoints
The exploratory endpoints are changes in the following parameters from baseline (V1) to post-intervention (V2): anaerobic capacity, haemoglobin mass and total blood volume, telomere length, systemic markers of inflammation, systemic coenzyme Q10, per- and polyfluoroalkyl substances (PFAS), RPE during exercise training and exercise capacity testing, and plasma metabolomics and proteomics. Finally, a broader proteome coverage (explorative non-defined outcomes) will be conducted which may yield insights into other potentially differentially regulated muscle properties in response to exercise and/or statin treatment.
Sample size calculations
The sample size was calculated to detect changes in the primary outcome, skeletal muscle CS maximal activity, and the key secondary outcome, peak oxygen uptake, between both exercise groups, comparable to previous exercise training studies conducted by our research group.55
Sample size calculation for CS maximal activity
Skeletal muscle CS activity is a widely used biomarker for skeletal muscle mitochondrial density and oxidative capacity,56 57 and a strong correlation exists between training-induced changes in CS maximal activity and changes in whole-body oxidative capacity.56 However, the clinically relevant effect size for changes in CS maximal activity is not clearly defined, and comparing values between studies is difficult due to different laboratories using varying methods and units of measurement.57 Furthermore, CS activity values measured in the same units can reportedly vary by up to a factor of 103 between studies.56 57 Thus, the present sample size calculations are based on data from a previous exercise training study conducted in our research group,55 which demonstrated that 8 weeks of endurance-based exercise training alone induced changes in CS maximal activity of 24.9 µmol/g/min with an SD of 18.2 µmol/g/min in middle-aged Faroese men and women. Based on our previous exercise training study, we expect the change in CS maximal activity to be normally distributed with an SD of 18.2 µmol/g/min. Thus, with a power of 0.80 and a significance level of 0.05 and assuming that statins treatment strongly mitigates the training-induced effect on CS maximal activity,13 the study requires 20 participants in each study arm to detect a difference of 16.9 µmol/g/min between groups. We will need 22 participants in each arm to attain a power of 0.85 and 26 completers in each arm to attain a statistical power of 0.90. With 60 enrolled study participants allocated to the exercise conditions and an expected dropout rate of 5%–10% following randomisation,58 59 a minimum of 27 participants from both exercise groups are expected to complete the intervention.
Sample size calculation for peak oxygen uptake
Aerobic capacity, determined as whole-body VO2peak, is a robust predictor of all-cause mortality and cardiovascular disease events,11 but statin treatment has been shown to completely abolish the training-induced effects on VO2peak in overweight participants.13 Therefore, peak oxygen uptake was selected as a key secondary outcome in the present study. Based on data from a previous exercise training study conducted in our research group,55 we estimated that 12 weeks of endurance-based exercise training alone would increase VO2peak by 3.6 mL/kg/min with an SD of 3.3 mL/kg/min. An increased VO2peak of 3.5 mL/kg/min is linked to a 13% lower risk of all-cause mortality and a 15% lower risk of cardiovascular disease events in healthy individuals,11 but some studies have defined a change in VO2peak of 2.5 mL/kg/min as the minimal clinically important difference.60 Thus, a between-group difference in VO2peak equivalent to approximately 3.0 mL/kg/min is expected to be physiologically relevant. Based on our previous exercise training study, we expect the changes in VO2peak to be normally distributed with an SD of 3.3 mL/kg/min. With a power of 0.80 and a significance level of 0.05, the study requires 21 participants in each study arm to detect a difference of 3.0 mL/kg/min between groups, while 23 participants in each arm are needed to attain a statistical power of 0.85 and 27 participants to attain a statistical power of 0.90.
Data collection, management and analysis
Clinical investigations
Study participants will attend identical clinical investigations before (baseline/V1) and after 12 weeks of treatment (end/V2) (figure 1). The clinical investigation is composed of two visits to the laboratory, each lasting approximately 2 hours. Table 1 provides a timeline for outcomes assessment.
Overview of study visits
Assessments criteria
Participants have fasted for ≥10 hours including foods, liquids and medication (not study medication) prior to all assessments.
Participants have taken their study medication on the morning of the tests.
Participants have refrained from vigorous exercise 48 hours before all assessments.
Alcohol, tobacco and caffeine have been avoided on assessment days.
Assessments
Anthropometrics
Total body weight (in kg with one decimal) will be obtained on a digital scale (InBody 270 multi-frequency body composition analyser; Biospace, California, USA) with light indoor clothing and without shoes. Abdominal waist circumference (the midpoint between the lowest rib and iliac crest) and hip circumference (the level of the great trochanters) will be measured in centimetres following a gentle expiration.
Skeletal muscle biopsies
Skeletal muscle biopsies will be taken under local anaesthesia (1% lidocaine) from the medial part of the m. vastus lateralis using the Bergstrom needle technique with suction.61 The muscle biopsy sample will be immediately frozen in liquid nitrogen and stored at −80°C until future analysis.
After freeze-drying, skeletal muscle biopsies will be analysed for maximal CS, 3-hydroxy-acetyl-CoA-dehydrogenase and phosphofructokinase activity, muscle fibre capillarisation, measurement of atorvastatin and coenzyme Q10, and for expression of complexes I–V. Also, muscle antioxidative capacity will be measured by determining the protein expression of superoxide dismutase 1 and 2.
For proteomic analyses, muscle lysates will be prepared by powdering of snap-frozen muscle biopsies (approx. 50 mg) before resuspending in lysis buffer (1% sodium deoxycholate, 50 mM Tris PH 8.5), followed by homogenisation using a tissue homogeniser (IKA turrax). Samples will then be analysed with data-independent parallel accumulation serial fragmentation mode on an Evosep One LC-system (Evosep, Denmark), in-line connected to a timsTOF SCP (Bruker). Data will be analysed in the DIA-NN software (V.1.8.1)62 followed by bioinformatic analysis via RStudio (differential abundance analysis, gene set enrichment analysis).
Exercise capacity
Three aspects of exercise capacity will be evaluated in the present study. First, VO2peak will be determined by an incremental cycling protocol to exhaustion on an electromagnetically braked cycling ergometer (Excalibur Sport, Lode, Groningen, Netherlands). Oxygen consumption (Cosmed, Quark b2, Milan, Italy) and heart rate (HRM-Dual, Garmin, Olathe, Kansas, USA) will be measured continuously throughout the test. Following a standardised 10-min warm-up protocol, the workload will be increased by 20 W/min (women) or 25 W/min (men) until peak oxygen consumption is attained in accordance with the previous criteria.63 The Borg 6-to-20 scale will be used <1 min after the warm-up protocol and again after the VO2peak test to assess the rate of perceived exertion during submaximal and maximal efforts. Second, anaerobic capacity will be assessed by a 30 s Wingate Anaerobic test with a fixed resistance of 0.075 kg per kg of total body mass for both men and women as previously described.64 Third, maximal strength will be determined as voluntary maximal isometric contraction force of the non-dominant arm using a JAMAR hand dynamometer (Performance Health) as previously described.59
Fasting blood samples
Fasting blood samples will be collected from the antecubital vein to measure bloodborne markers of metabolic health (including lipid profile, insulin, blood glucose, c-peptide and HbA1c), coenzyme Q10, telomere length, plasma proteomics and metabolomics as well as markers of plasma inflammation (including interferon gamma, interleukin (IL)-10, IL-2, IL-6, IL-8, IL-1beta, IL-1 receptor antagonist, tumor necrosis factor alpha, C-reactive protein and leukocytes) and PFAS. For participants’ safety, blood samples will also be analysed for haemoglobin, thrombocytes, creatinine, estimated glomerular filtration rate, creatine kinase, alanine-aminotransferase, bilirubin, alkaline phosphatase, thyroid stimulating hormone, albumin and electrolytes (sodium and potassium).
Furthermore, a blood sample for genetic analysis of candidate genes involved in the transport, metabolism and clearance of statins will be collected in a 3 mL EDTA vacuum container. Specifically, the polymorphisms of the genes encoding the uptake transporter SLCO1B1, the effluent transporters ABCB1, ABCC2, ABCG2, the apolipoproteins APOE, APOA5 and the cytochrome P450 enzyme system including KIF6, HMGCR, LDLR, LPA, PCSK9, COQ2, CETP will be analysed.44
Body composition
Total body fat and lean tissue will be assessed using dual-energy X-ray absorptiometry (DEXA) (Norland XR-800, Norland Corporation).
Haemoglobin mass and blood volume
Haemoglobin mass and blood volume will be measured by the carbon monoxide rebreathing method as described in detail previously.65
Blood pressure
Blood pressure and resting heart rate will be measured using a digital blood pressure monitor. The average of three measurements from the left upper arm in a sitting position will be taken after at least 5 min of rest.66
Questionnaires
Questionnaires related to physical activity (IPAQ-SF)67 and quality of life (SF-36v2)68 will be completed at baseline and post-intervention. 10 health concepts ranging from 0 to 100 will be scored from the SF-36v2 questionnaire: physical functioning, role limitations due to physical health problems, bodily pain, general health perception, vitality (energy/fatigue), social functioning, role limitations due to emotional problems, mental well-being and physical and mental component summary. Furthermore, questionnaires related to SAMS46 will be asked frequently throughout the intervention.
Data management
Study participants will only be identified through their assigned study ID (pseudonymisation). The REDCap electronic data capture tool,69 which will be hosted by the National Hospital of the Faroe Islands, will be used by the study personnel to collect and manage study data. Data extraction procedures will be undertaken by the investigators. Sharing data between the University of the Faroe Islands and international collaborators will be controlled through data agreement contracts. All obtained biological material will be labelled with study ID and stored in a safe research biobank at the National Hospital of the Faroe Islands. To access and use biological material, which has not been used for the original study analysis, a new protocol must be approved by the ethics committee and informed consent must be obtained from the study participants. The trial has been registered in the Faroese Data Protection Agency and complies with the Data Protection Act.
Data analysis plan
All statistical analyses will be pre-specified in a Statistical Analysis Plan finalised prior to unblinding. Analyses will be performed using a validated statistical software package (eg, SPSS, R, SAS or Stata). Two-sided p values <0.05 will be considered statistically significant unless otherwise stated. Because multiple outcome measures are planned, we will also report effect sizes (eg, mean differences with 95% CIs) and, where appropriate, correct for multiple comparisons to guard against type I error inflation.
Our primary linear mixed‐effects models (LMMs) will include fixed effects for group, time and group×time interaction, with random intercepts for participants. Stratification factors (sex and statin use) will be included as covariates in the model as well as baseline adjustment for the corresponding outcomes.70 71
Analysis of population and handling of missing data
Analyses will be performed according to both:
Intention-to-treat (ITT) principle: includes all participants randomised, regardless of adherence or dropout.
Per-protocol principle: includes participants who meet both of the following minimum adherence requirements:
Exercise adherence: ≥75% of the prescribed interval-training time performed at ≥90% of maximal aerobic power (ie, the lowest cycling power output (W) that elicits V̇O₂max).
Medication adherence: ≥75% adherence to the allocated medication regimen.
In the ITT analysis, missing data will be handled implicitly by maximum likelihood estimation in the mixed model under the missing at random assumption. Sensitivity analyses (eg, complete case analysis, pattern-mixture models) will assess the robustness of conclusions to different missing data mechanisms.
Baseline comparisons
Participant characteristics will be summarised by the intervention arm at baseline. For continuous variables, means (SD) or medians (IQR) will be reported; for categorical variables, counts (%) will be presented.
Primary outcome analysis
The primary endpoint is the change in skeletal muscle CS maximal activity µmol/g/min from baseline (V1) to the end of the intervention (V2). We will fit an LMM to the repeated measurements, with fixed effects for the group (four levels), time (baseline vs post-intervention) and the interaction of group×time. This model will include a random intercept for each participant to account for within-subject correlation. If model diagnostics (eg, residual plots) indicate skewness or heteroscedasticity, the outcome may be log-transformed. Model fit will be assessed via visual inspection of residuals.
From this model, we will obtain:
Between-group differences in the change from baseline to post-intervention (primary contrast: exercise+placebo vs exercise+atorvastatin, and additional contrasts among the four arms).
Within-group changes (baseline to post-intervention) for each group.
95% CIs for each difference and change.
All estimates from the primary outcome will be reported with effect sizes (mean differences or ratios, depending on transformation).
Secondary outcome analysis
Each continuous secondary endpoint will be analysed using a similar LMM framework, with group, time and their interaction as fixed effects, and random intercepts for participants. Categorical endpoints (eg, presence/absence of SAMS if binarised) will be assessed using logistic regression.
Exploratory and proteomic analyses
Exploratory endpoints (eg, anaerobic capacity, telomerase activity, inflammatory markers, plasma metabolomics, muscle and plasma proteomics) will be analysed in an exploratory framework, where effect sizes and 95% CIs will be emphasised. For untargeted proteomics, raw files will be processed via state-of-the-art pipelines followed by R-based analyses.
Genetic and interaction analysis
Candidate gene polymorphisms and their potential interaction with exercise or statin allocation will be investigated using either:
Mixed-effects models incorporating genotype (or allele count) and its interaction with group×time.
Add-on analyses assessing how participant subgroups (eg, carriers vs non-carriers of a specific polymorphism) differ in primary or secondary outcomes.
Given the increased likelihood of multiple comparisons in genetics analyses, results will be interpreted cautiously, applying false discovery rate corrections where necessary.
Sex-specific analysis
We will also evaluate whether sex modifies the intervention effects by including a three-way interaction term (group×time×sex) in the mixed-effects models. This approach will help identify any sex-specific differences in metabolic responses, statin-associated effects or exercise adaptations, providing a more nuanced understanding of treatment efficacy across patient subgroups.
Interim analysis and data monitoring
No interim analyses for efficacy are planned, given the relatively short duration and the aim of this exploratory, mechanistic trial. A Data Monitoring and Safety Committee (DMSC) will periodically review participant safety data (eg, adverse events). Unblinding will occur only under exceptional clinical circumstances or as required by the DMSC for serious adverse event management.
Reporting
Study findings for primary, secondary and exploratory outcomes will be reported according to the CONSORT (Consolidated Standards of Reporting Trials) guidelines. Both statistical significance (via p values) and clinical relevance (via effect sizes and CIs) will be clearly presented. All major analyses will be documented in the final report, including null or negative findings.
Rationale for the four-group design
A four-arm design was selected to isolate and compare the individual and combined effects of exercise and atorvastatin (80 mg/day) on mitochondrial function and other health-related outcomes in individuals with dyslipidaemia. Specifically:
Exercise+placebo: serves as the exercise-only group, enabling us to evaluate the beneficial adaptations attributable solely to exercise training.
Exercise+atorvastatin (80 mg/day): assesses whether statin therapy and exercise together produce additive or antagonistic effects on mitochondrial function and aerobic capacity compared with exercise alone or atorvastatin alone. This group also allows us to determine whether exercise can counteract any potential decline in mitochondrial function or other health parameters that might be observed in the atorvastatin-only group.
Atorvastatin (80 mg/day): evaluates the effects of high-dose statin therapy in the absence of exercise, thus distinguishing medication-specific effects from those induced by physical activity. Having this group also clarifies whether any deleterious changes—particularly a decline in mitochondrial function—can be mitigated when exercise is added.
Placebo: serves as the no-treatment control, enabling comparisons with participants receiving either statins, exercise or both. Including this group is crucial for maintaining blinding in the atorvastatin (80 mg/day) group; without a placebo control, participants would know they are receiving the active medication, potentially biasing their behaviour and/or expectations. The placebo group also controls for improvements unrelated to the specific interventions, such as changes in diet or lifestyle prompted simply by participation in the study.
By comparing these four groups, we can:
Parse out the separate effects of exercise and high-dose atorvastatin.
Examine potential synergy or antagonism when both interventions are combined.
Interpret any observed changes relative to a true no-treatment control.
This factorial approach is integral for understanding how exercise and statins independently and jointly influence key outcomes such as skeletal muscle mitochondrial function, aerobic fitness and metabolic markers. It also provides a robust framework to assess whether combining statin therapy with exercise training can ameliorate or prevent any negative impact of statins on mitochondrial function or other health outcomes.
Discussion
The present research protocol is the first randomised, double-blinded, placebo-controlled trial in middle-aged individuals with dyslipidaemia to investigate the combined effects of statins and exercise training on a broad range of health markers, including skeletal muscle, cardiovascular and blood-based adaptations, as well as quality of life. By employing untargeted proteomic analyses, this study aims to identify novel biomarkers and molecular pathways involved in exercise-induced and statin-induced changes, potentially leading to more targeted therapeutic approaches and better-informed clinical recommendations for managing dyslipidaemia. The integration of genetic analysis in this study may further identify genetic polymorphisms that influence individual responses to statins and exercise, paving the way for personalised treatment strategies based on genetic predispositions.
Despite its strengths, the study has some limitations. First, the exclusion of patients with established ASCVD, while beneficial for isolating the effects of statins and exercise in a population with dyslipidaemia without significant cardiovascular conditions, may limit the generalisability of the findings to a broader, high-risk population. Future research should aim to include patients with ASCVD to evaluate how these interventions affect those with more advanced cardiovascular conditions. Additionally, the 12-week duration of the study may not capture the long-term physiological adaptations or adverse effects associated with prolonged statin use and exercise. Longer-term follow-up studies are needed to provide a comprehensive understanding of the statin–exercise interaction over time.
In summary, this research proposal seeks to address a critical gap in the understanding of how statins and exercise interact at the molecular level, using advanced techniques such as proteomic analysis and genetic profiling. The findings could significantly impact clinical practice by optimising treatment strategies for individuals with dyslipidaemia, ultimately improving their ability to engage in high-intensity exercise without experiencing adverse muscle symptoms and enhancing overall health outcomes.
Ethics and dissemination
Medication safety and monitoring
Participants will be administered atorvastatin at a dose of 80 mg/day, which is the maximum approved maintenance dosage.72 While atorvastatin is widely used and generally considered safe, it is associated with potential myopathies, including muscle aches, tenderness and weakness, with creatine phosphokinase levels potentially reaching up to ten times the upper limit of normal.73 Other common adverse effects include arthralgia, dyspepsia, diarrhoea, nausea, nasopharyngitis, insomnia and urinary tract infections.73 To ensure participant safety, liver function tests will be conducted at baseline. Given that patients with ischaemic heart disease are excluded, there is no known risk to the life and well-being of the study participants. Participants will be closely monitored by a cardiologist throughout the intervention period. The inclusion of a placebo group is ethically justified as it allows for the isolation of the specific effects of exercise and statins. Any increase in LDL-C levels due to statin withdrawal in the placebo groups will be managed according to predefined safety protocols.
Exercise training considerations
The prescribed exercise intervention is designed to be intense but manageable through a gradual increase in session intensity and frequency. The exercise protocol consists of non-weight-bearing activities, minimising the risk of exercise-induced injuries and ensuring safe participation for all subjects.
Risk management for physiological assessments
The risks associated with physiological assessments are deemed minimal and are outweighed by the potential benefits. Specific risks include:
Muscle biopsies: risk of minor bleeding, local soreness (2–3 weeks) and rare cases of temporary sensory nerve damage (1 in 2783 cases).74
Blood sampling: mild bruising or discomfort lasting up to 2 weeks.
Exercise testing: discomfort similar to that experienced during strenuous exercise, mitigated by pre-screening for cardiovascular risk.
DEXA scans: during DEXA scans, participants will be exposed to a modest radiation dose of approximately 0.02 mSv per scan.
We have previously conducted similar training studies at the Faroe Islands that also included extensive physiological assessments in both healthy individuals55 59 and patients.58 75 76 All interventions were well-tolerated by participants, as expected. Overall, the risks associated with the present study are considered minimal, and the benefits of participating are expected to significantly outweigh the potential risks.
Ethical considerations
Although there are no direct clinical benefits from participating in the study, participants will receive close health monitoring throughout the intervention period and valuable feedback on their fitness and metabolic health. This study aims to yield new insights into how statins and exercise interact at the molecular level, potentially refining treatment strategies for individuals with dyslipidaemia and enabling them to engage in high-intensity exercise without adverse muscle symptoms, ultimately promoting improved overall health outcomes. The study will be conducted in compliance with the Declaration of Helsinki, and written informed consent will be obtained from all participants before any study-related activities begin.
Dissemination of results
The findings from this study, whether positive or negative, will be published in accordance with the CONSORT 2010 guidelines77 in international peer-reviewed journals. Study results will also be presented at scientific conferences to maximise the impact and dissemination of the findings within the academic and clinical communities. All participants will receive a lay summary of the study’s main results.
Ethics statements
Patient consent for publication
References
Footnotes
Contributors TS formulated, initiated and designed this study. TS, SL, SBKJ, JB, JR, HE, JFV-L, NN and MM contributed to the overall study design. TS, SL, SBKJ and MM contributed to the detailed description of the study interventions, assessments and data analysis plan. TS drafted the manuscript. TS, SL, SBKJ, JB, JR, SB, JK, JMM, HWO, HE, JFV-L, NN and MM have contributed to and approved the final version of the manuscript. Authorship eligibility follows the Vancouver guidelines. TS is the guarantor and accepts full responsibility for the finished work and/or the conduct of the study, has access to the data and controls the decision to publish. We used AI-based language processing tools solely to perform grammatical checks and minor stylistic adjustments on the text. These tools did not alter the scientific content, methodology or conclusions presented in the manuscript. The technology served strictly as a proofreading aid and did not generate or modify the substantive content of the submission.
Funding This work was supported by the Research Council Faroe Islands (Granskingarráðið) grant number (0360) to cover some salaries, operating expenses and analyses, and by Betri Stuðul to cover some running operating expenses and analyses.
Competing interests The planning and conduct of the study, interpretation of data and writing of manuscripts are completely independent of the funders.
Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.