When using 13C or 14C tracers to study substrate metabolism, an acetate correction factor should be applied to correct for loss of label in the exchange pathways of the tricarboxylic acid cycle. We have shown recently that the [13C]acetate recovery factor has a high inter-individual variability and should therefore be determined in every subject. In the present study we examined the factors that might explain some of the variability between subjects in acetate recovery factor. Data were pooled from four different studies with identical protocols, in which the acetate recovery factor was measured, prior to an intervention, to correct plasma fatty acid oxidation rates. Acetate recovery was measured after 2 h of [1,2-13C]acetate infusion at rest followed by 1 h of cycling exercise at 40–50% of maximal oxygen uptake. Inter-individual variance in acetate recovery was 12.0% at rest and 16.1% during exercise. Stepwise regression revealed that, at rest, 37.1% of the acetate recovery could be accounted for by basal metabolic rate adjusted for fat-free mass, percentage body fat and respiratory quotient (RQ). During exercise, 69.1% of the variance in acetate recovery could be accounted for by energy expenditure adjusted for fat-free mass, % body fat and RQ. In conclusion, we show that the acetate recovery factor has a high inter-individual variability, both at rest and during exercise, which can partly be accounted for by metabolic rate, RQ and % body fat. These data indicate that the acetate recovery factor needs to be determined in every subject, under similar conditions as used for the tracer-derived determination of substrate oxidation. Failure to do this might result in large under- or over-estimation of plasma substrate oxidation, and hence to artificial differences between groups.