The VetScan® i-STAT® 1 Handheld Analyzer and cardiac troponin I (cTnI) cartridges (i-STAT cTnI assay) measured greater median cTnI concentration [cTnI] in free-ranging white-tailed deer (Odocoileus virginianus) hand-injected with anesthetic drugs after physical restraint in Clover traps than in those ground-darted with the same drugs. This suggested that Clover trapping induces myocardial damage, bringing the use of this capture method under scrutiny. The purpose of this study was to confirm the validity of the i-STAT cTnI assay in deer before recommending changes in capture methods. Median [cTnI] measured by the i-STAT cTnI assay ([cTnI]_i) in heparinized whole blood collected from 52 healthy, reproductively mature, female deer physically restrained in a chute was 0.01 ng/ml (10–90% percentiles: 0.00–0.03 ng/ml; minimum, maximum: 0.00, 0.07 ng/ml); [cTnI]_i was 0.00 ng/ml in 42% of the deer. There was no association between [cTnI]_i and either clotting or hemolytic index. [cTnI]_i was 0.00 ng/ml when deer skeletal muscle homogenate was added to deer blood with [cTnI]_i of 0.00 ng/ml, confirming the i-STAT cTnI assay does not detect skeletal muscle troponins. When deer cardiac muscle homogenate was serially diluted with 1) deer blood, 2) deer plasma, and 3) cow blood, [cTnI]_i was directly proportional (Y intercept = −0.09, 0.7, and −0.08 ng/ml, respectively; r² ≥ 0.97) to the fraction of homogenate in each sample. Deer cardiac muscle homogenate was diluted with deer blood to produce three samples with low, intermediate, and high [cTnI]_i; serial measurements (n = 10) performed on each sample yielded coefficients of variation (CVs) of 8, 20, and 11%, respectively. Corresponding CVs when plasma was used as diluent were 13, 9, and 7%, respectively. [cTnI]_i increased when plasma with a low [cTnI]_i was stored at 20–24°C for 9 days. Three freeze–thaw cycles caused no systematic change in plasma [cTnI]_i.