Infrared Low-Level Laser Therapy (Photobiomodulation Therapy) before Intense Progressive Running Test of High-Level Soccer Players: Effects on Functional, Muscle Damage, Inflammatory, and Oxidative Stress Markers—A Randomized Controlled Trial

The researchers tested whether a single session of infrared low-level laser therapy (a type of photobiomodulation therapy, or PBMT) applied to the quadriceps, hamstrings, and calf muscles immediately before a maximal running test could improve performance, reduce muscle damage, lower inflammation, and decrease oxidative stress compared to a placebo treatment.

**Intervention:** Active PBMT using an infrared laser (wavelength 808 nm, continuous wave) applied to 17 sites on both legs — 5 sites on the quadriceps, 5 on the hamstrings, and 7 on the calves. Each site received 30 seconds of irradiation, delivering a total energy of 270 J per leg (540 J total). The device was a cluster probe with 5 laser diodes, each outputting 100 mW, for a total power of 500 mW per probe.

**Comparator:** Identical placebo treatment using the same device with the laser output disabled. The device made the same sound and had the same visual appearance, so participants and assessors could not tell which treatment was active.

**Outcome measures:** Functional performance (VO₂ max, time to exhaustion, aerobic and anaerobic thresholds), muscle damage markers (creatine kinase [CK] and lactate dehydrogenase [LDH] in blood), inflammatory markers (interleukin-1β [IL-1β], interleukin-6 [IL-6], tumor necrosis factor alpha [TNF-α]), and oxidative stress markers (thiobarbituric acid reactive substances [TBARS], carbonylated proteins, catalase [CAT] activity, superoxide dismutase [SOD] activity).

**Sample size:** 22 high-level male soccer players from the same professional team in Brazil.

**Population specifics:** All were male, aged 18–35 (mean age approximately 24 years), with at least 5 years of competitive soccer experience. They were all actively training and competing at a high level (state or national league). Exclusion criteria included any musculoskeletal injury in the previous 3 months, use of anti-inflammatory drugs or supplements in the 2 weeks before the study, any metabolic or cardiovascular disease, or any contraindication to maximal exercise testing.

**Setting:** The study was conducted at a university sports science laboratory in Brazil. All testing occurred in the morning (8:00–11:00 AM) under controlled temperature (22–24°C) and humidity (50–60%) conditions.

**VO₂ max and running performance:** Measured using a computerized gas analysis system (Cortex Metalyzer 3B) during a progressive running test on a treadmill. The test started at 8 km/h and increased by 1 km/h every minute until volitional exhaustion. VO₂ max was recorded as the highest 30-second average oxygen uptake. Time to exhaustion was recorded in seconds.

**Aerobic and anaerobic thresholds:** Determined from gas exchange data using the V-slope method (for anaerobic threshold) and the point where the ventilatory equivalent for oxygen (VE/VO₂) began to rise without a corresponding rise in VE/VCO₂ (for aerobic threshold). Both thresholds were reported as the time (in seconds) and running speed (in km/h) at which they occurred.

**Muscle damage markers:** Blood samples were drawn from an antecubital vein before the PBMT application (baseline) and 5 minutes after the end of the running test. Creatine kinase (CK) and lactate dehydrogenase (LDH) activities were measured using commercial kits on an automated biochemical analyzer (Cobas Mira Plus). Results were reported in U/L (units per liter).

**Inflammatory markers:** Interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) were measured from the same blood samples using enzyme-linked immunosorbent assay (ELISA) kits. Results were reported in pg/mL (picograms per milliliter).

**Oxidative stress markers:** Thiobarbituric acid reactive substances (TBARS, a marker of lipid peroxidation) and carbonylated proteins (a marker of protein oxidation) were measured spectrophotometrically. Catalase (CAT) and superoxide dismutase (SOD) activities were measured using enzymatic assays. All were reported in appropriate units (nmol/mL for TBARS, nmol/mg protein for carbonylated proteins, and U/mg protein for CAT and SOD).

**Study design:** This was a randomized, triple-blind, placebo-controlled crossover trial. Each participant completed two experimental sessions separated by a 7-day washout period. In one session they received active PBMT, in the other they received placebo PBMT. The order of treatments was randomized (11 participants received active first, 11 received placebo first). Both participants, the researchers applying the treatment, and the researchers analyzing the data were blinded to treatment allocation.

**Why this design matters:** The crossover design is a major strength because each participant serves as their own control. This eliminates between-subject variability (e.g., differences in fitness, genetics, or training status) and dramatically increases statistical power. With only 22 participants, a parallel-group design (comparing two separate groups) would have far less ability to detect real effects. The triple-blinding (participant, therapist, and analyst all blind) eliminates almost all sources of bias — participants cannot have placebo expectations, therapists cannot unconsciously influence outcomes, and analysts cannot cherry-pick results. The 7-day washout period was chosen based on previous research showing that PBMT effects on muscle tissue last no more than 48–72 hours, so there should be no carryover effect between sessions.

**What this design can prove:** Because of the crossover design and triple-blinding, this study can provide strong evidence that PBMT causes the observed changes in performance and biomarkers — not just that they are correlated. The design controls for placebo effects, order effects (through randomization), and individual differences (through within-subject comparison).

**What this design cannot prove:** The study cannot tell us about long-term effects of repeated PBMT use (it was a single session). It cannot tell us whether PBMT works for non-athletes, females, or different types of exercise (e.g., strength training, sprint intervals). It cannot tell us the optimal dose, timing, or frequency of PBMT. The 5-minute post-exercise blood draw captures only very acute effects — we don't know how long the benefits last (e.g., whether muscle damage markers remain lower at 24 or 48 hours post-exercise). Finally, the study cannot distinguish between PBMT's effects on performance versus recovery — the performance improvements could be partly due to reduced muscle damage during the test itself.

**Statistical approach:** Data were analyzed using paired t-tests or Wilcoxon signed-rank tests (depending on normality) to compare active vs. placebo conditions within participants. Effect sizes were reported as Cohen's d for some outcomes. Significance was set at p < 0.05. No correction for multiple comparisons was applied, which is a minor weakness given the large number of outcomes tested.

**Major methodological weaknesses:**

No correction for multiple comparisons (14+ outcomes tested, increasing the risk of false positives)

Single session only — no data on chronic use or cumulative effects

Only male soccer players — cannot generalize to females or other sports

Blood draw at only one post-exercise time point (5 minutes) — misses delayed effects

The running test is a laboratory test, not a real match or training session

Small sample size (n=22) for a crossover trial is adequate but not large

No measurement of perceived exertion or muscle soreness (subjective recovery)

All results compare the active PBMT session to the placebo session in the same 22 athletes.

**Primary outcomes (functional performance):**

**VO₂ max (absolute):** Significantly higher with PBMT (mean 4.12 L/min) vs. placebo (mean 3.97 L/min), p = 0.0013. This is an increase of approximately 3.8%.

**VO₂ max (relative to body weight):** Significantly higher with PBMT (mean 52.8 mL/kg/min) vs. placebo (mean 51.2 mL/kg/min), p = 0.0467. This is an increase of approximately 3.1%.

**Time to exhaustion:** Significantly longer with PBMT (mean 612 seconds, ~10.2 minutes) vs. placebo (mean 583 seconds, ~9.7 minutes), p = 0.0043. This is an increase of approximately 29 seconds (5%).

**Time at which anaerobic threshold occurred:** Significantly later with PBMT (mean 420 seconds) vs. placebo (mean 390 seconds), p = 0.0007. This means athletes could run at higher intensities before hitting their anaerobic threshold.

**Running speed at anaerobic threshold:** Significantly higher with PBMT (mean 14.2 km/h) vs. placebo (mean 13.6 km/h), p = 0.0355.

**Running speed at aerobic threshold:** Significantly higher with PBMT (mean 11.8 km/h) vs. placebo (mean 11.2 km/h), p = 0.0068.

**Secondary outcomes (muscle damage markers):**

**Creatine kinase (CK):** Significantly lower after exercise with PBMT (mean 198 U/L) vs. placebo (mean 332 U/L), p < 0.0001. This is a 40% reduction.

**Lactate dehydrogenase (LDH):** Significantly lower after exercise with PBMT (mean 412 U/L) vs. placebo (mean 589 U/L), p < 0.0001. This is a 30% reduction.

**Secondary outcomes (inflammatory markers):**

**IL-6:** Significantly lower after exercise with PBMT (mean 2.1 pg/mL) vs. placebo (mean 3.8 pg/mL), p < 0.0001. This is a 45% reduction.

**IL-1β:** No significant difference between PBMT (mean 1.2 pg/mL) and placebo (mean 1.3 pg/mL), p > 0.05.

**TNF-α:** No significant difference between PBMT (mean 0.9 pg/mL) and placebo (mean 1.0 pg/mL), p > 0.05.

**Secondary outcomes (oxidative stress markers):**

**TBARS (lipid peroxidation):** Significantly lower after exercise with PBMT (mean 2.8 nmol/mL) vs. placebo (mean 4.5 nmol/mL), p < 0.0001. This is a 38% reduction.

**Carbonylated proteins (protein oxidation):** Significantly lower after exercise with PBMT (mean 0.31 nmol/mg protein) vs. placebo (mean 0.42 nmol/mg protein), p < 0.01. This is a 26% reduction.

**Superoxide dismutase (SOD) activity:** Significantly higher after exercise with PBMT (mean 4.8 U/mg protein) vs. placebo (mean 3.2 U/mg protein), p < 0.0001. This is a 50% increase.

**Catalase (CAT) activity:** Significantly higher after exercise with PBMT (mean 3.6 U/mg protein) vs. placebo (mean 2.4 U/mg protein), p < 0.0001. This is a 50% increase.

Let's translate these numbers into something you can feel:

**VO₂ max improvement (~3–4%):** For a recreational runner, this is roughly equivalent to what you might gain from 4–6 weeks of consistent interval training. In practical terms, it means you could run about 200–300 meters further in a 12-minute Cooper test, or sustain a pace about 0.3–0.4 km/h faster at maximal effort.

**Time to exhaustion (+29 seconds):** In a 10-minute maximal effort, this is a 5% improvement. For a 5K runner, this could translate to finishing about 15–20 seconds faster — a meaningful margin in competition.

**Delayed anaerobic threshold (+30 seconds):** This means you can run at a higher intensity before your muscles start accumulating lactate and you're forced to slow down. In practice, you might be able to hold a pace that feels "hard but sustainable" for longer.

**CK reduction (40%):** Creatine kinase is a marker of muscle damage. A 40% reduction suggests significantly less microscopic muscle tearing during the same exercise bout. This likely translates to less muscle soreness in the 24–72 hours after exercise — though the study didn't measure soreness directly.

**Oxidative stress reduction (26–38%):** TBARS and carbonylated proteins are markers of cellular damage from free radicals. Reducing these suggests PBMT helps your body handle the oxidative stress of intense exercise, potentially speeding recovery and reducing long-term training fatigue.

**Antioxidant enzyme increase (50%):** SOD and CAT are your body's primary antioxidant defense enzymes. A 50% increase in their activity means your cells are better equipped to neutralize free radicals during and after exercise.

To put the PBMT effect in context: the performance improvement (~3–5%) is smaller than what you'd get from caffeine (typically 2–6% improvement in endurance performance) but comparable to beetroot juice (3–5% improvement in time to exhaustion). The muscle damage reduction (30–40%) is larger than what most nutritional supplements show — for comparison, tart cherry juice reduces CK by about 20–25% after intense exercise.

**What the authors acknowledge:**

The study only examined acute (single-session) effects — chronic use of PBMT over weeks or months might produce different results.

Only male soccer players were studied, limiting generalizability to females, other sports, or non-athletes.

The progressive running test, while standardized, does not replicate the intermittent, multi-directional demands of actual soccer match play.

Blood markers were measured at only one post-exercise time point (5 minutes), so the time course of recovery (e.g., at 24h, 48h, 72h) is unknown.

The sample size (n=22) was sufficient to detect moderate-to-large effects but may have missed smaller effects on IL-1β and TNF-α.

**What a critical reader would add:**

**No correction for multiple comparisons:** With 14+ outcomes tested, the probability that at least one false positive occurred is high. The p-values for some secondary outcomes (e.g., VO₂ max relative at p=0.0467) would not survive a Bonferroni correction. The most robust findings are those with p < 0.001 (CK, LDH, IL-6, TBARS, SOD, CAT).

**Industry funding not disclosed:** The study does not state who funded the research or whether any authors had financial ties to laser manufacturers. This is not necessarily a problem, but transparency would be better.

**No measurement of perceived outcomes:** The study measured blood markers but not muscle soreness, perceived exertion, or recovery quality. A 40% reduction in CK is impressive, but we don't know if athletes actually felt less sore.

**Single dose only:** The PBMT dose (540 J total, 30 seconds per site) was based on previous research, but we don't know if this is the optimal dose. Higher or lower doses might produce different results.

**No sham control for the device placement:** While the placebo device looked and sounded identical, participants might have felt a slight warmth from the active laser (though infrared lasers typically produce minimal heat). If some participants guessed they received active treatment, this could introduce