This study was a randomised, double-blind, placebo-controlled cross-over trial examining the effects of β-hydroxy β-methylbutyrate free acid (HMB-FA) supplementation on muscle protein breakdown, cortisol, testosterone and resting energy expenditure (REE) during acute fasting. Conditions consisted of supplementation with 3 g/d HMB-FA or placebo during a 3-d meat-free diet followed by a 24-h fast. Urine was collected before and during the 24-h fast for analysis of 3-methylhistidine:creatinine ratio (3MH:CR). Salivary cortisol, testosterone, their ratio (T:C), and the cortisol awakening response were assessed. ANOVA was used to analyse all dependent variables, and linear mixed models were used to confirm the absence of carryover effects. Eleven participants (six females, five males) completed the study. Urinary HMB concentrations confirmed compliance with supplementation. 3MH:CR was unaffected by fasting and supplementation, but the cortisol awakening response differed between conditions. In both conditions, cortisol increased from awakening to 30 min post-awakening (P=0·01). Cortisol was reduced from 30 to 45 min post-awakening with HMB-FA (−32 %, d=−1·0, P=0·04), but not placebo (PL) (−6 %, d=−0·2, P=0·14). In males, T:C increased from 0 to 24 h of fasting with HMB-FA (+162 %, d=3·0, P=0·001), but not placebo (+13 %, d=0·4, P=0·60), due to reductions in cortisol. REE was higher at 24 h of fasting than 16 h of fasting independent of supplementation (+4·0 %, d=0·3, P=0·04). In conclusion, HMB-FA may affect cortisol responses, but not myofibrillar proteolysis, during acute 24-h fasting.