Background - Digit ratio (2D:4D) is a putative marker for prenatal testosterone and is correlated with performance in many sports. Low 2D:4D has been linked to strength but the evidence is mixed and strength is also influenced by mass, testosterone, and behavioural factors. It has been hypothesised that the 2D:4D-strength correlation may be strongest in challenge conditions when short-term changes occur in steroid hormones. Results - In the challenge condition there was a highly significant increase in HGS, and modest changes in T, physical aggression and emotional stability. HGS correlated negatively with left hand 2D:4D. In a multiple regression, left hand 2D:4D was negatively related to HGS and emotional stability was positively related to HGS. In the control condition HGS was not correlated with 2D:4D. In a multiple regression, BMI, physical aggression, and emotional stability were significantly related to HGS. Conclusions - 2D:4D is a negative correlate of strength in challenge situations. This finding may in part explain associations between 2D:4D and sports performance.