Heat stress (HS) causes alterations in blood biomarkers, however, limited research is available on the effects of rumen-protected methionine (RPM) feeding or sample harvesting time on blood biomarkers during HS. Thirty-two multiparous, lactating Holstein cows [DIM (184 ± 59)] were randomly assigned to 1 of 2 environmental treatment groups, and 1 of 2 dietary treatments [TMR with RPM (Smartamine M; Adisseo Inc., Antony, France; 0.105% DM of TMR as top dress) or TMR without RPM (CON)] in a crossover design. The study was divided into 2 periods with 2 phases per period. During phase 1 (9d), all cows were in thermoneutral conditions (TN; THI = 60 ± 3) and fed ad libitum. During phase 2 (9d), group 1 (n = 16) was exposed to HS using electric heat blankets. Group 2 (n = 16) remained in TN (THI = 61 ± 4) but was pair-fed (PFTN) to HS counterparts. After a 21-d washout period, the study was repeated (period 2). Environmental treatments were inverted relative phase 2 in period 1, while the dietary treatments remained the same. Plasma samples were taken 4 and 8 h post-feeding on d 1, 3, 6, and 9 of each phase, and phase 1 samples from each period were pooled relative to h collected. Paired difference values were calculated for each cow for each period based on the difference between phase 1 (baseline) values and phase 2 values for each variable at each time point (4 or 8 h). Statistical analysis was performed on paired difference values using MIXED procedure of SAS. At 4 h, cows in RPM tended to have greater (P = 0.10) increase in plasma urea nitrogen (PUN) concentration for PFTN (+1.26 mg/dL) and HS (+1.08 mg/dL) compared with cows in CON for PFTN (+0.68 mg/dL) and HS (+0.15 mg/dL). At 4 h, PUN concentration change was not different between cows in PFTN and HS (+0.97 vs. +0.62 mg/dL, respectively; P = 0.31). However, at 8 h, cows in PFTN had greater decrease in PUN compared with cows in HS (−0.66 mg/dL vs. −0.09 mg/dL, respectively; P = 0.04). At 8 h, cows in PFTN had a greater increase in NEFA concentration compared with cows in HS (+9.61 vs. +0.77 µEg/L; P = 0.04). In conclusion, HS altered blood biomarker concentration compared with PFTN. Additionally, blood sample timing effected biomarker concentration.