临床试验雷达

Serum butyrate concentrations

Diet and in particular fiber intake will be standardized for three days prior to and during the assessment days during which venous blood sample collection will take place. To promote microbial SCFAs production, participants will consume 15 g of oligofructose five hours prior to venous blood sample collection. Serum butyrate concentration will be assessed using gas chromatography-mass spectrometry.

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Fecal butyrate concentrations

Diet and in particular fiber intake will be standardized for three days prior to and during the assessment days during which fecal collection will take place. Participants will be asked to collect a stool sample at the first experiment assessment day. Fecal butyrate concentration will be assessed using gas chromatography with flame ionization detection.

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Short-chain fatty acids

Short-chain fatty acid concentration (i.e. acetate, propionate, and butyrate concentrations) will be assessed in serum (gas chromatography-mass spectrometry) and feces (gas chromatography with flame ionization detection).

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Gut microbiome

The gut microbiome will be evaluated using taxonomic composition and functional genetic capacity (shotgun metagenomics, feces).

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Gut barrier

Indices of gut barrier integrity will be assessed by lactulose mannitol ratio test (HPLC-ELSD) and plasma lipopolysaccharide binding protein (ELISA).

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Self-reported stool consistency, bowel movement frequency, and gastrointestinal distress

A gastrointestinal questionnaire will be used to report stool consistency, bowel movement frequency, and gastrointestinal distress.

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Aerobic exercise performance

Aerobic exercise performance will be measured on a bicycle ergometer through a 30-min simulated time-trial test.

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Anaerobic exercise performance

Anaerobic exercise performance will be measured on a bicycle ergometer through a 6-s isokinetic sprint test, and a 30-s isokinetic sprint test.

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Cardiorespiratory fitness

Cardiorespiratory fitness will be evaluated through an incremental maximal cardiopulmonary exercise test (step test), throughout which peak oxygen uptake and the first and second lactate thresholds will be determined

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Knee extensor strength

Knee extensor strength will be assessed using a custom-build dynamometer.

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Muscle fiber type composition

Muscle fiber type composition will be visualized and quantified using fluorescent immunohistochemical staining.

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Muscular mitochondrial content, biogenesis, and remodelling

Markers of mitochondrial content, biogenesis, and remodelling will be quantified using a hierarchical analytical workflow combining western blotting, enzyme histochemistry, and spectrophotometric assays.

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Glucose tolerance and insulin sensitivity in skeletal muscle

If the oral glucose tolerance test indicates between-group differences in glucose tolerance or insulin sensitivity, skeletal muscle samples will undergo western blot analysis to quantify key insulin-signalling proteins.

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Heart rate

Heart rate will be measured using a Polar Pacer monitor connected to a Polar H10 chest strap. Resting heart rate will be assessed once weekly, while submaximal and maximal heart rate will be continuously recorded during all training and testing sessions.

Each training session, once every weekend, and at day 2 and day 3 of each experimental assessment period.

Heart rate variability

Heart rate variability (HRV) will be measured using a Polar Pacer monitor connected to a Polar H10 chest strap. RR intervals for HRV will be collected once weekly and during each experimental assessment timepoint, with measurements performed immediately upon waking in a supine position. HRV will be quantified using Kubios software, including time-domain, frequency-domain, and non-linear indices.

Each training session, once every weekend, and at day 2 and day 3 of each experimental assessment period.

Inflammatory status

Changes in serum hsCRP will be evaluated as marker of systemic low-grade inflammation. Gut-local inflammation will be assessed via fecal calprotectin. In case differential inflammatory or maladaptive responses are observed between study arms, a predefined subset of stored biosamples will be subjected to more detailed inflammatory and gut-barrier profiling within available resources. If moderate vs high load training induces distinct changes in intestinal or systemic inflammatory status, gene and protein expression levels of muscle pro- and anti-inflammatory cytokines will be analyzed using commercially available ELISAs

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Hormonal status

Concentrations of resting and exercise-induced hormones related to the hypothalamic-pituitary-adrenal (HPA) axis, stress, energy regulation, and cortisol will be analyzed in venous blood and saliva samples using electrochemiluminescence immunoassay (ECLIA) or enzyme-linked immunosorbent assay (ELISA).

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Self-reported recovery-stress state

Participants will complete the validated Recovery-Stress Questionnaire for Athletes (RESTQ-Sport) to assess perceived recovery-stress balance. The RESTQ-Sport comprises 77 items across 19 scales. Each item is rated on a 7-point Likert scale (0 = "never" to 6 = "always"). The recovery-stress balance will be calculated by subtracting the total stress score (sum of 10 stress subscales) from the total recovery score (sum of 9 recovery subscales), with higher values indicating more favourable recovery states.

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Self-reported mood disturbances

Mood disturbance will be assessed using the validated Profile of Mood States (POMS) questionnaire (McNair et al., 1971). The POMS is a 65-item instrument that evaluates transient, fluctuating mood states. It comprises six subscales: five negative affect dimensions (Fatigue, Depression, Tension, Anger, and Confusion) and one positive affect dimension (Vigour). Participants rate how they have felt during the past week on a 5-point Likert scale (0 = "not at all" to 4 = "extremely"). The total mood disturbance (TMD) score will be calculated by summing the five negative subscales and subtracting the positive (Vigour) subscale, with higher TMD values indicating greater overall mood disturbance. An energy index will be calculated as the difference between the POMS Vigour and Fatigue subscale scores, providing an indicator of perceived energy and readiness to perform.

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Self-reported physical and mental work load

Subjective workload will be assessed using the validated NASA Task Load Index (NASA-TLX) questionnaire, a reliable indicator of both physical and mental workload (Sandra \& Lowell, 1988). The NASA-TLX comprises six dimensions: Mental Demand, Physical Demand, Temporal Demand, Frustration, Effort, and Performance. Participants will rate each dimension on a 20-point scale ranging from "very low" to "very high." The overall workload score will be calculated as the mean of the six dimension ratings, with higher scores indicating greater perceived workload.

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Self-reported physical and mental well-being

Subjective well-being will be assessed using the Wellness Questionnaire (Bellinger, 2020), which comprises seven items evaluating physical and mental well-being. Participants will rate each item on a 1-10 Likert scale, where higher scores indicate better perceived well-being. The questionnaire will be completed at the end of each intervention week and at each experimental assessment period. An overall well-being score will be calculated as the sum of all item scores.

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Self-reported symptoms of upper respiratory tract infections

The Wisconsin Upper Respiratory Symptom Survey (WURSS-21) will be used to monitor the incidence and severity of upper respiratory tract infections (Barrett et al., 2009). Participants will rate one global question, ten symptom-related questions, and nine functional-impairment questions (e.g., ability to think, sleep, breathe, walk) on a 0-7 Likert scale (0 = no symptoms, 1 = very mild, 3 = mild, 5 = moderate, 7 = severe), based on average symptom intensity over the previous seven days. The total WURSS-21 score will be calculated as the sum of all items, with a score ≥ 21 indicating the incidence of an upper respiratory tract infection (URTI).

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Vascular health

Peripheral microvascular function will be assessed using a vascular occlusion test combined with near-infrared spectroscopy on the gastrocnemius muscle. The protocol will consist of a 2-min baseline recording, 8 min of arterial occlusion, and 5 min of reperfusion. Muscle oxygen extraction will be quantified from the StO₂ decline during occlusion, and microvascular reperfusion capacity will be determined from the rate of StO₂ recovery after cuff release.

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

HOMA-IR

A venous blood sample will be collected after an overnight fast to analyze fasting glucose and insulin concentrations. Fasting glucose levels and the homeostasis model assessment of insulin resistance (HOMA-IR) will serve as indicators of liver insulin resistance.

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Matsuda index

The Matsuda index, derived from an oral glucose tolerance test, will be used as a marker for muscle and whole-body insulin resistance. In this test, 75 g of glucose will be administered following a 10-12 hour overnight fast, and venous glucose and insulin levels will be measured at 0, 30, 60, 90, and 120 minutes post-administration.

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Body composition

Body composition will be measured by a Dual Energy X-ray Absorptiometry (DXA) scan.

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Food intake

Diet and nutritional intake will be monitored using a food diary (MijnEetmeter) during three days each week. Additionally, participants will follow a standardized dietary protocol for three days before and during the experimental assessment periods.

During three days each week during the study and three days before and during the experimental assessment periods.

Muscle protein synthesis and breakdown

If DXA reveals between-group changes in whole-body or regional fat-free mass, western blotting will be used to assess regulators of muscle protein synthesis and breakdown.

At baseline (T1), mid-intervention after four intervention weeks (T2), post-intervention after eight intervention weeks (T3), and 10 days following intervention completion (T4).

Intervention adherence and training monitoring

Adherence and fidelity to the training intervention will be assessed by recording the number of completed sessions, and the sessions' power output, heart rate, and session duration. Adherence to the dietary supplementation in the control group will be ensured via directly observed administration. Continuation of participants' habitual sport activities outside the intervention will be monitored using the Polar Pacer smartwatch.

From baseline (T1) until 10 days following intervention completion (T4).

Number of participants classified as overreached

Participants in the high-load training group will be classified at T3 as overreached when both of the following are present relative to T2: (i) a reduction in exercise performance, defined as a lower mean power output during either the 30-second sprint test or the 30-minute time trial, and (ii) a reduction in the energy index calculated from the POMS questionnaire as Vigour minus Fatigue. To account for day-to-day variability, the reduction in exercise performance must exceed the coefficient of variation derived from the control group, and the reduction in energy index must exceed the standard error of measurement of the change score derived from the control group. Participants showing a reduction in energy index without a decrement in exercise performance will be classified as acutely fatigued.

Post-intervention (T3, week 8)