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Macaluso, F, Morici, G, Catanese, P, Ardizzone, NM, Marino Gammazza, A, Bonsignore, G, Giudice, GL, Stampone, T, Barone, R, Farina, F, and Di Felice, V. Effect of conjugated linoleic acid on testosterone levels in vitro and in vivo after an acute bout of resistance exercise. J Strength Cond Res 26(6): 1667–1674, 2012—The purposes of the present study were to investigate the effect of conjugated linoleic acid (CLA) supplementation on testosterone levels in vitro on a cell line derived from Leydig cells (R2C) and in vivo in the blood of physically active subjects before and after a resistance exercise bout. In vitro R2C cells were treated with different CLA concentrations (0–30 μM) for 24 and 48 hours. After treatment, supernatant media were tested to determine testosterone secretion. The CLA increased the testosterone secretion only after 48 hours. In vivo, 10 resistance-trained male subjects, in a double-blind placebo-controlled and crossover study design were randomized for 3 weeks of either 6 g·d−1 CLA or placebo. Blood was drawn pre and post each resistance exercise bout to determine the total testosterone and sex hormone–binding globulin (SHBG) levels. No significant differences were observed for total testosterone or SHBG pre and post each resistance exercise bout; although after the resistance exercise bouts, total testosterone increased moderately (effect size = moderate), whereas after CLA supplementation, there was a large increase in total testosterone (effect size = large). CLA supplementation induced an increase in testosterone levels in Leydig cells in vitro after 48 hours but not in vivo before and after a resistance exercise bout. These findings suggest that CLA supplementation may promote testosterone synthesis through a molecular pathway that should be investigated in the future, although this effect did not have an anabolic relevance in our in vivo model.