The objective of this study was to evaluate short-term variations of trans fatty acids (TFA) in plasma lipoproteins and ruminal fermentation parameters of non-lactating cows subjected to ruminal pulses of vegetable oils. Three non-lactating, non-pregnant Holstein cows, each with a ruminal cannula, were arranged in a 3 × 3 Latin square design with three-day pulsing periods and four-day washout intervals between treatments. Cows were treated with single ruminal pulses of: (1) control (skimmed milk (SM); 500 mL); (2) soybean oil (SO; 250 g/d in 500 mL of SM) and (3) partially-hydrogenated vegetable oil (PHVO; 250 g/d in 500 mL of SM). Time changes after infusion in TFA contents were only observed for plasma C18:1 trans-4, trans-5 and trans-12, and high-density lipoprotein fraction C18:1 trans-9. After ruminal pulses, concentration of acetate decreased linearly; molar concentrations of propionate and valerate increased linearly; molar concentrations of butyrate and isovalerate changed quadratically and were greater at 1 h than at other times. There was an accumulation of several C18:1 TFA in plasma and lipoproteins, especially on the third day of pulsing. Overall, naturally occurring C18:1 TFA isomers (produced during ruminal biohydrogenation of SO) and preformed TFA (supplied by PHVO) elicited differential TFA partitioning and transport in plasma and lipoproteins.