Ethical aspects

The study protocol was submitted to and approved by the Ethics Committee of the Federal Rural University of Rio de Janeiro under protocol No. 01/2025. All procedures were conducted in accordance with the ethical principles established by the National Council for the Control of Animal Experimentation (CONCEA) and complied with current legislation.

Sample

Initially, twenty-one male Wistar Albino rats (Rattus norvegicus), 13 weeks old (91 days) and weighing 331 ± 20 g, were used in this study. An a priori sample size calculation was performed using G*Power software (version 3.1.9.7) for a one-way ANOVA. To comply with the ethical principle of Reduction (3Rs), the calculation predicted that a total of 21 animals (n = 7 per group) would be sufficient to detect a large effect size (f = 0.751) with a statistical power of 0.80 and a significance level of α = 0.05.

Animals were randomly assigned to three experimental groups: Trained (TR, n = 7), Trained + nimesulide (COMB, n = 7), and Control (CTRL, n = 7). However, due to unforeseen mortality unrelated to the experimental procedures, three animals from the CTRL group were lost during the protocol. To maintain temporal synchronization and avoid confounding variables associated with a new experimental cohort, these animals were not replaced, resulting in a final sample of n = 4 for the CTRL group. Statistical analyses for unbalanced designs were applied to ensure the validity and viability of the results.

Experimental design

The study was conducted in two stages. The first stage consisted of a seven-day adaptation period to familiarize the animals with the procedures. Animals were randomized in a blinded manner. All groups were habituated to the resistance training ladder and gavage administration to minimize stress-related effects. Animals were housed under controlled conditions (22 °C; 12:12 h light-dark cycle) with ad libitum access to food and water.

The second stage involved pre- and post-training strength assessments in the trained and control groups. The training protocol lasted eight weeks, with one session every three days (totaling 20 sessions) (Fig. 1A).

Fig. 1

Methodological design and exercise training apparatus. (A) Experimental study design. (B) Ladder-based resistance training apparatus

Drug preparation and administration

Nimesulide (Cimed®, Brazil) was administered at a therapeutic dose of 2.5 mg/kg (50 mg/mL) (Dhir et al. 2007; Saghaei et al. 2012). A 1:20 dilution was prepared by mixing 1 mL of the stock solution with 19 mL of 0.9% saline. The solution was stirred magnetically (INTLAB™) and administered via orogastric gavage after each training session throughout the eight-week period. To ensure dosage accuracy, body weight was monitored weekly and doses were adjusted accordingly. The treated group received the drug, while the control and trained groups received 0.9% saline to control the stress effects associated with gavage.

Familiarization with the resistance training apparatus

The resistance exercise apparatus consisted of a ladder measuring 110 cm in length, with a housing chamber at the top and an entry hole at the base (Fig. 1B). Prior to performing the maximum load test, the animals underwent a familiarization period with the experimental apparatus. After that, the animals performed three ladder climbs per session over seven consecutive days, carrying tail-attached fabric pouches containing adjustable lead weights for each training session. The pouch featured two straps that were fixed at the proximal portion of the animals’ tails.

Maximum capacity test

Following Hornberger and Farrar (2004), animals began the test with a load corresponding to 75% of their body weight (BW). Load increments of 30 g (~ 10 ± 2% BW) were added until failure to complete the climb. The maximum load successfully lifted was recorded as the maximum carrying capacity. Tests were performed in the 1 st and 20th sessions.

Resistance training protocol

Starting at 91 days of age, the TR animals underwent ladder-climbing resistance training three times per week for eight weeks. Each session consisted of 4–9 climbs with progressively increasing loads (50%, 75%, 90%, and 100% of maximum capacity), with 2-minute rest intervals. Subsequent climbs added 30 g until a new maximum was achieved. Thus, the rats’ new maximum load capacity was calculated in each training session. This regimen was repeated every three days for eight weeks, totaling 20 training sessions (Hornberger and Farrar 2004).

Volume load

Volume load is a quantitative measure of the total work performed in an exercise, muscle group, or resistance training session. It combines volume (number of sets and repetitions) with intensity (weight lifted). It is calculated as the product of the number of sets, the number of repetitions, and the load used (in kilograms or pounds). This variable is used to more comprehensively quantify the training stimulus, prescribe and periodize training to allow progressive adjustments, monitor overload over time as an indicator of progression or fatigue, and compare workload across exercises, sessions, or training cycles (Fleck and Kraemer 2014, p. 6; Krause Neto et al. 2018).

Euthanasia and post-mortem analysis

Within three days of completing the final session, animals were euthanized by decapitation. The Flexor Hallucis Longus (FHL) muscle was excised, rinsed in 0.9% saline, weighed, and stored at − 80 °C for future histological and biomolecular analyses.

Statistical analysis

Normality was assessed using the Shapiro–Wilk test. Body mass, maximum absolute load, and maximum normalized load were analyzed using two-way ANOVA (group × time), with repeated measures on the time factor, followed by appropriate post hoc tests. Tukey’s multiple comparisons test was applied for between-group comparisons, while Bonferroni corrections were used for within-group comparisons across time, as appropriate. Volume load across training sessions was analyzed using two-way ANOVA followed by Tukey’s post hoc test. FHL hypertrophy was analyzed using one-way between-groups ANOVA followed by Tukey’s post hoc test. Statistical significance was set at p ≤ 0.05. Data are presented as mean ± standard error of the mean (SEM) and were analyzed using GraphPad Prism version 9.0 (GraphPad Software, CA, USA).