Twelve healthy elite Israeli junior handball players (age range 17–20 years) participated in the study. All the participants played in the Israeli premier handball league, and some of the players belonged to the Israeli national junior handball team. The study was performed during the final stages of the regular handball season when the players are at their best physical shape. Training in this part of the season involved mainly tactic and technical drills emphasizing handball skills and team strategies, speed drills with and without the ball, and longer interval sessions (e.g., several repetitions of 20- to 40-s run at ~80% of the maximal speed). No resistance training was done at the time period of the study. The study was approved by the Institutional Review Board of the Meir General Hospital, and informed consent was obtained from all the participants.
Anthropometric characteristics of the participants are summarized in Table 1. Standard, calibrated scales, and stadiometer were used to determine height and body mass, and BMI was calculated from these data. Skinfold measurement at four sites (triceps, biceps, sub-scapular, and supra-iliac) was used to estimate percent body fat using standard equations (Slaughter et al. 1988).
Exercise and cryotherapy protocol
Participants were instructed to avoid any physical activity, caffeine, and/or alcohol use at the day before each study. None of the participants used medications and/or dietary supplements at the time of the study.
Each participant performed twice an outdoors maximal 100-m run. The best result was used to calculate the speed of the interval exercise session. Exercise consisted of four 250-m runs on a treadmill (motor-driven treadmill; Woodway, PPS 55med, Weil am Rhein, Germany), at a constant intensity of 80% of the maximal speed (calculated from the speed of 100-m run), with 3-min rest between each of the 250-m runs. This protocol was performed twice at random order, once without and once with local cold-pack application following the interval practice. The cold packs (half size 5 inch × 12 inch Whitehall Glacier-Packs™ Reusable Cold Packs) were applied immediately following the final 250-m run using a compression wrap. The cold packs were applied to the hamstring muscles for 15 min, followed by 15-min interval, and then again for additional 15 min while the athletes were resting supine in a quiet room. Room temperature was 21°C. Following the session without cold-pack application, the athletes rested in the same conditions for 1 h.
Blood sampling and analysis
Tests were performed in the morning, following an overnight fast. An indwelling venous catheter was inserted 30 min prior to the first blood draw. Pre, immediately after the last 250-m run, and 60-min post the last 250-m run (recovery) blood samples were drawn from the catheter (Fig. 1). Blood samples were immediately spun at 3,000 rpm, at 4°C for 20 min. The serum was separated and stored at −80°C. All pre- and post-exercise specimens from each individual were analyzed in the same batch by an experienced technician who was blinded to the order of samples and to the type of interval training.
Growth hormone serum concentrations were determined by ELISA with the use of the DSL-10-1900 Active kit (Diagnostic System Laboratories, Webster, Texas). Intra-assay CV was 3.3–4.5%, inter-assay CV was 5.5–12.9%, and the sensitivity was 0.03 ng/ml.
Insulin-like growth factor-I
Insulin-like growth factor-I was extracted from IGF-binding proteins (IGFBPs) by using the acid-ethanol extraction method. Serum IGF-I concentrations were determined by a two-site immunoradiometric assay by using the DSL-5600 Active kit (Diagnostic System Laboratories, Webster, TX). IGF-I intra-assay CV was 1.5–3.4% and the inter-assay CV was 3.7–8.2%. Assay sensitivity was 0.8 ng/ml.
IGF-binding proteins-1 was measured by a coated-tube immunoradiometric assay with the use of the DSL-10-7800 Active kit (Diagnostic System Laboratories). Intra-assay CV was 2–4%, and inter-assay CV was 1.7–6.7%. Assay sensitivity is 0.33 ng/ml. IGFBP-3 serum concentrations were determined by ELISA with the use of the DSL 10-6600 Active kit (Diagnostic System Laboratories). Intra-assay CV was 7.3–9.6%, inter-assay CV was 8.2–11.4%, and the sensitivity was 0.04 ng/ml.
Serum cortisol levels were determined by a commercial RIA (Diagnostic Products Corporation, Los Angeles, CA). The intra- and inter-assay CV were 3.2 and 6.8%, respectively.
Testosterone serum concentrations were determined by ELISA with the use of the DSL commercial kit (Diagnostic System Laboratories, Webster, Texas). Intra-assay CV was 4.8–5.3%, inter-assay CV was 2.8–4.9%, and the sensitivity was 0.04 ng/ml.
Inflammatory mediators were analyzed by ELISA, using the R&D system Quantikine High Sensitivity commercial kits (R&D system; Minneapolis, MN).
Intra-assay CV was 3.8–11.1%, inter-assay CV was 7.1–29.5%, and the sensitivity was 0.0094 pg/ml.
Interleukin-1 beta (IL-1β)
Intra-assay CV was 1.6–4.0%, inter-assay CV was 5.3–9.0%, and the sensitivity was 0.059 pg/ml.
Interleukin-1 receptor antagonist (IL-1ra)
Intra-assay CV was 3.1–6.2%, inter-assay CV was 4.4–6.7%, and the sensitivity was 22 pg/ml.
Intra-assay CV was 8.1–15.6%, inter-assay CV was 6.6–8.2%, and the sensitivity was 0.5 pg/ml.
A two-way repeated measure ANOVA (with Bonferroni post hoc test) was used to assess the effect of exercise on circulating components of the GH–IGF-I axis and inflammatory mediators with time serving as the within group factor and post-exercise cold-pack application as the between group factor. Data are presented as mean ± SEM. Significance was taken at P ≤ 0.05.