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Completed

NCT Number: NCT07771075

High-Intensity Interval Training and Monocyte Epigenetic Adaptation After Detraining

This randomized, work-matched training-detraining-rechallenge study evaluated whether repeated high-intensity interval training produces persistent functional and epigenetic adaptations in human monocytes. Eighty healthy physically inactive adults aged 18-30 years were randomly assigned in a 2:2:1 ratio to high-intensity interval training (HIIT), moderate-intensity continuous training (MCT), or a non-training control group. HIIT and MCT completed 6 weeks of supervised cycling training matched for external mechanical work, followed by 4 weeks without prescribed structured training. All groups subsequently completed a standardized 30-minute cycling rechallenge. The primary outcome was the between-group difference in change in ex-vivo LPS-stimulated TNF-alpha release from baseline to the post-detraining assessment. Secondary outcomes included IL-6 release, H3K18 lactylation, monocyte subset distribution, blood lactate exposure, chromatin accessibility, H3K18la CUT&Tag occupancy, and RNA-seq transcriptional profiling.

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Key information

Conditions

Physical Inactivity Behavior Sedentary Behavior

Age range

18 year–30 year

Sex eligibility

All sexes

Study type

Interventional

Phase

Not applicable

Primary location

Exercise Physiology Laboratory, College of Physical Education and Sports Sciences, University of Samarra

Samarra, Muhafazat Salah ad Din, Iraq

About this study

Participants completed baseline cardiopulmonary exercise testing to determine peak oxygen uptake (VO2peak) and maximal aerobic power (MAP). Eligible participants were randomized to HIIT, MCT, or Control. The HIIT intervention consisted of 4 × 4-minute cycling intervals at 85% MAP separated by active recovery at 40% MAP, with standardized warm-up and cool-down periods. The MCT intervention consisted of continuous cycling at 55% MAP for a duration selected to match the external mechanical work of the HIIT sessions. Both exercise groups trained three times per week for 6 weeks.

Blood and monocyte assessments were performed at baseline (T0), 72 hours after the final training session (T1), after 4 weeks without prescribed structured training (T2_PRE), and 120 minutes after a standardized 30-minute cycling rechallenge at 60% of baseline MAP (T3_POST).

Classical monocytes were isolated and stimulated ex vivo with ultrapure Escherichia coli O111:B4 lipopolysaccharide (10 ng/mL) for 24 hours. TNF-alpha was the prespecified primary functional outcome, with IL-6 as a key secondary outcome. Epigenetic and molecular analyses included global H3K18 lactylation, ATAC-seq chromatin accessibility profiling, H3K18la CUT&Tag, and RNA-seq. Blood lactate kinetics and cumulative lactate exposure across the training intervention were also assessed.

The primary comparison was HIIT versus MCT. The Control group served as a supportive non-training reference. Longitudinal outcomes were analyzed using mixed models for repeated measures under the intention-to-treat principle.

Who can participate

Healthy volunteers accepted: Yes

Only the study team can determine whether someone qualifies for participation.

Inclusion

Men and women aged 18-30 years. Healthy adults. Physically inactive, defined as participating in less than 150 minutes per week of structured moderate-to-vigorous exercise during the preceding 6 months.

Exclusion criteria

  • Major cardiovascular, metabolic, respiratory, renal, endocrine, inflammatory, autoimmune, or hematological disease.

Current tobacco use. Current use of relevant immunomodulatory, anti-inflammatory, corticosteroid, or cardiovascular medications.

Acute infection within 4 weeks before baseline assessment. Musculoskeletal contraindications to maximal cycling exercise. Pregnancy or lactation.

Treatment and study plan

High-intensity interval training

Behavioral

Supervised cycling HIIT performed three sessions per week for 6 weeks. Each session included a 5-minute warm-up at 30% MAP, four 4-minute intervals at 85% MAP separated by three 3-minute active recovery periods at 40% MAP, and a 5-minute cool-down at 30% MAP.

Moderate-Intensity Continuous Training

Behavioral

Supervised moderate-intensity continuous cycling performed three sessions per week for 6 weeks. Each session included a 5-minute warm-up at 30% MAP, 31 minutes and 16 seconds of continuous cycling at 55% MAP, and a 5-minute cool-down at 30% MAP. The protocol was designed to match the external mechanical work performed by the HIIT group.

Primary outcomes

  1. Change in LPS-Stimulated TNF-Alpha Release From Classical Monocytes

    Time frame: Baseline (T0) and 4 weeks after completion of the 6-week training intervention (T2_PRE).

    TNF-alpha concentration (pg/mL) was measured in culture supernatants after 24-hour ex-vivo stimulation of purified classical CD14++CD16- monocytes with 10 ng/mL ultrapure Escherichia coli O111:B4 lipopolysaccharide. The primary outcome was the change from baseline (T0) to the post-detraining assessment (T2_PRE), with the primary comparison between the HIIT and MCT groups.

Secondary outcomes

  1. Change in LPS-Stimulated IL-6 Release From Classical Monocytes

    Time frame: Baseline (T0) and 4 weeks after completion of the 6-week training intervention (T2_PRE).

    IL-6 concentration (pg/mL) was measured in culture supernatants after 24-hour ex-vivo stimulation of purified classical CD14++CD16- monocytes with 10 ng/mL ultrapure Escherichia coli O111:B4 lipopolysaccharide. The outcome was the change from baseline (T0) to the post-detraining assessment (T2_PRE), with comparison between the HIIT and MCT groups.

  2. Exercise-Rechallenge Change in LPS-Stimulated TNF-Alpha Release From Classical Monocytes

    Time frame: Immediately before the standardized exercise rechallenge (T2_PRE) and 120 minutes after the rechallenge (T3_POST).

    TNF-alpha concentration (pg/mL) was measured after 24-hour ex-vivo stimulation of purified classical CD14++CD16- monocytes with 10 ng/mL ultrapure Escherichia coli O111:B4 lipopolysaccharide. The outcome was the within-participant change from the post-detraining pre-rechallenge assessment (T2_PRE) to 120 minutes after the standardized exercise rechallenge (T3_POST), with the principal comparison between the HIIT and MCT groups.

  3. Exercise-Rechallenge Change in LPS-Stimulated IL-6 Release From Classical Monocytes

    Time frame: Immediately before the standardized exercise rechallenge (T2_PRE) and 120 minutes after the rechallenge (T3_POST).

    IL-6 concentration (pg/mL) was measured after 24-hour ex-vivo stimulation of purified classical CD14++CD16- monocytes with 10 ng/mL ultrapure Escherichia coli O111:B4 lipopolysaccharide. The outcome was the within-participant change from the post-detraining pre-rechallenge assessment (T2_PRE) to 120 minutes after the standardized exercise rechallenge (T3_POST), with the principal comparison between the HIIT and MCT groups.

  4. Change in Monocyte H3K18 Lactylation Across Study Time Points

    Time frame: Baseline (T0), 72 hours after the final training session (T1), 4 weeks after training cessation before rechallenge (T2_PRE), and 120 minutes after the standardized exercise rechallenge (T3_POST).

    Histone H3 lysine 18 lactylation (H3K18la) was quantified in purified classical CD14++CD16- monocytes and normalized to total histone H3 abundance. H3K18la was assessed to determine training-induced elevation, persistence after 4 weeks without prescribed structured training, and re-amplification following the standardized exercise rechallenge.

  5. Genome-Wide Chromatin Accessibility in Classical Monocytes

    Time frame: Four weeks after completion of the 6-week training intervention, before the standardized exercise rechallenge (T2_PRE).

    Genome-wide chromatin accessibility was assessed by ATAC-seq in purified classical CD14++CD16- monocytes. Differentially accessible regions between the HIIT and MCT groups were identified using a false discovery rate (FDR) <0.05 and an absolute log2 fold-change >1.0.

  6. Genome-Wide H3K18la Occupancy in Classical Monocytes

    Time frame: Four weeks after completion of the 6-week training intervention, before the standardized exercise rechallenge (T2_PRE).

    Genome-wide histone H3 lysine 18 lactylation (H3K18la) occupancy was profiled by CUT&Tag in purified classical CD14++CD16- monocytes. H3K18la-enriched genomic regions were identified and their overlap with differentially accessible ATAC-seq regions was evaluated between the HIIT and MCT groups.

  7. Genome-Wide Differential Gene Expression in Classical Monocytes

    Time frame: Four weeks after completion of the 6-week training intervention, before the standardized exercise rechallenge (T2_PRE).

    Genome-wide transcript abundance was assessed by RNA sequencing (RNA-seq) in purified classical CD14++CD16- monocytes. Differential gene expression between the HIIT and MCT groups was evaluated using DESeq2, with differentially expressed genes defined using a false discovery rate (FDR) <0.05 and an absolute log2 fold-change >1.0.

Other outcomes

  1. Monocyte Subset Distribution Across Study Time Points

    Time frame: Baseline (T0), 72 hours after the final training session (T1), 4 weeks after training cessation before rechallenge (T2_PRE), and 120 minutes after the standardized exercise rechallenge (T3_POST).

    Peripheral blood monocyte subset distribution was quantified by flow cytometry as the percentage of classical (CD14++CD16-), intermediate (CD14++CD16+), and non-classical (CD14+CD16++) monocytes among viable HLA-DR+ monocytes. This outcome was used to evaluate whether training, detraining, or the standardized exercise rechallenge altered circulating monocyte subset composition.

  2. Blood Lactate Exposure During the 6-Week Training Intervention

    Time frame: Across the 18 supervised training sessions during the 6-week intervention.

    Blood lactate exposure was assessed during the supervised HIIT and MCT training interventions. Measures included acute peak blood lactate concentration, session lactate area under the concentration-time curve (AUC), and cumulative lactate AUC across the 18 training sessions. Lactate exposure was compared between the work-matched HIIT and MCT groups.

Sponsors and collaborators

Lead sponsor

Saif Rasheed Ghanim

Other

Registry information

Official study title

Repeated High-Intensity Exercise Induces Persistent H3K18 Lactylation and Trained Immunity in Human Monocytes: A Randomized Work-Matched Training-Detraining-Rechallenge Trial

Important dates

Study start
2026
Primary completion
2026
Study completion
2026
First posted
Aug 18, 2026
Registry last updated
Aug 18, 2026

OpenTrials presents study information sourced from ClinicalTrials.gov. The official registry record should be consulted for the latest information.

View the official ClinicalTrials.gov record (opens in a new tab)

This listing is for discovery and informational purposes only. It is not medical advice, does not guarantee that a study is recruiting, and does not determine eligibility. Contact the study team and a qualified healthcare professional when considering participation.

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