Participants were recruited from among the 72 soccer players who were invited to the Swiss Soccer Association’s national selection day. One of this study’s authors (the project leader) invited all 72 players to participate in the study. The cross-sectional 2D:4D sample included 63 (87.5%) male soccer players who decided to participate in the study. All of the participants were in good health and free of acute or known chronic diseases at the time of the study. The study was approved by the local research ethics committees (Kantonale Ethikkommission Bern, Switzerland, No. 022/13). Written informed consent was obtained from all of the parents and each of the participants. The participants were informed that participation was voluntary and that they could withdraw from the study at any time. Weight, height, and 2D:4D were measured. To measure weight, the participants wore shorts and a T-shirt. Height was measured with a stadiometer (Seca 217; Seca, Hamburg, Germany), and weight was measured with calibrated scales (Tanita WB-110 MA; Tanita, Tokyo, Japan). Weight and height were measured to the nearest 0.1 kg and 0.1 cm, respectively. All of the hand-wrist X-rays and DXA scans were performed at the Swiss Olympic Medical Center Magglingen according to hand-wrist guidelines. With the participants sitting beside the X-ray device (Stadler SE 4600; Stadler, Littau, Switzerland), the left hand-wrist was placed without any radial or ulnar deviance on a double-layered phosphor cassette. Using this standardization, posterior-anterior radiographs of the left hand were taken with an X-ray device. All digital X-ray scans were analysed on a computer screen using iQ-VIEW 2.5.0 (IMAGE Information Systems, London, UK). A standardized modus of 42 kV tube voltage and 1.60 mAs, with a radiation time of 0.78 s, was used. Subsequently, on the same day, each participant underwent a DXA scan of the left hand-wrist (iDXA; General Electric Lunar, 2008, Madison, WI). Scans were analysed using software supplied by GE Medical Systems (GE enCORE 2011, version 13.60). This software allows a left hand wrist scan which were performed with all participants. Automatic brightness and contrast optimisation was used. All the scans were executed by one investigator using a standardized modus of 100 kV tube voltage and 0.19 mAs. With the participants sitting beside the DXA device, the scan was focused on the hand-wrist, starting four centimeters below the radiocarpal joint in order to obtain an image of the distal radius, the wrist, and all of the hand bones. All of the X-ray and DXA images were saved without any participant characteristics in order to insure that all of the analyses were blind assessments. All of the scans were analyzed by two independent, trained raters (R1, R2). The measurements were made with precision clear rulers to the nearest of 0.5 mm. The distances of 2D and 4D were measured from the base of the proximal phalanx to the tip of the distal phalanx [
30]. R1 and R2 independently assessed each of the participants’ 2D:4Ds on the X-ray and the DXA scan to evaluate interrater variability, and they analyzed these a second time four weeks later to evaluate intrarater reliability. The assessments were repeated after four weeks in order to minimize recall bias. Residuals were examined for normality, linearity. The normality assumption was checked using Kolmogorov-Smirnov (KS) test and linearity with lack of fit test. To compare the two techniques employed to assess 2D:4D, we used the statistical methods described by Bland and Altman [
31]. Intrarater and interrater reliability were analyzed using intraclass correlation coefficients (ICCs) with a 95% confidence interval (CI). Values of less than 0.40 indicated poor reliability, values of 0.40-0.60 indicated fair reliability, values of 0.60-0.75 indicated good reliability, and values greater than 0.75 indicated excellent reliability [
32]. Bland-Altman plots were used to visualize the differences between the X-rays and the DXA scans and their distribution. The mean 2D:4D, the mean difference, the standard deviation (SD) of the mean difference, 95% limits of agreement (LoA), and standard error of the estimate (SEE) were calculated. In accordance with previous validation studies, we decided to accept that the mean difference between the two techniques could deviate by a maximum of 5% from the mean of both techniques and to accept that the LoA could be within a range of ±0.05. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) version 21.0 (IBM SPSS, Chicago, Illinois, USA). The level of significance was set at P < 0.05.