Variation of the method for determining the enzyme hydrolysate gross energy (EHGE) of feedstuff using in vitro bionic digestion was investigated to confirm the precision of assay and evaluate the factors contributed to the variation of determined values. A single factorial completely randomized design was adapted. Three computer-controlled simulated digestion system (SDS-2) were randomly selected to determine the dry matter digestibility (DMD) and EHGE in corn, wheat, rapeseed meal and cottonseed meal. Each feedstuff was repeatedly determined for the DMD and EHGE in 3 batches. Each batch contained 5 replicates with 1 digestion tube per replicate. The variation coefficients of variables for determining the DMD and EHGE were calculated and the correlation between these variables and DMD or EHGE were developed. The results showed as follows: 1) the intra-batch, inter-batch and total coefficient of variation (CV) were all less than 1.00% for DMD and EHGE in corn, wheat and cottonseed meal. The intra-batch, inter-batch and total CV were all less than 1.00% for DMD and 1.64% for EHGE in rapeseed meal, respectively. In bionic digestion, the intra-batch, inter-batch and total CV were all less than 0.40%, 4.16%, 4.17% and 2.08% for sample weight, residues weight, residues energy and residues energy content, respectively. 2) Correlation between sample weight and DMD was not significant for corn, wheat and rapeseed meal (P>0.05), also low for cottonseed meal (|r|0.05). However, high correlation between residues weight and DMD, and between residues energy and EHGE were observed for all feedstuffs (|r|>0.95, P0.05). In conclusion, the precision is acceptable for the method to determine DMD and EHGE of feedstuffs using bionic digestion based on SDS-2. The variation of residues weight is the main factor contributed to the variation of DMD and EHGE.