<PAGE>   1
                                                                    Exhibit 10.1

                               THIRD AMENDMENT TO
                           LOAN AND SECURITY AGREEMENT

         THIS THIRD AMENDMENT TO LOAN AND SECURITY AGREEMENT (the "Amendment")
is made this the 23rd day of March, 2001 by and among SUNTRUST BANK (formerly
SunTrust Bank, Nashville, N.A.) (the "Lender") and LANDAIR CORPORATION and
LANDAIR TRANSPORT, INC. (collectively, the "Borrower").

                                    RECITALS:

         A. Borrower and Lender entered into that certain Loan and Security
Agreement dated January 5, 1999, as amended by a First Amendment to Loan and
Security Agreement and as amended by a Second Amendment to Loan and Security
Agreement dated August 26, 1999 (as amended from time to time, the "Agreement").

         B. The Borrower and the Lender are desirous of amending the Agreement
as set forth below.

         C. Terms not defined herein shall have the meanings ascribed to such
terms in the Agreement.

         NOW, THEREFORE, in consideration of the premises and mutual covenants
and obligations herein, the parties hereto agree that the Agreement is hereby
amended and modified as follows:

         Section 1. Section 1.4 of the Agreement concerning "Interest" is
amended to the extent that the Applicable Margin shall be one and one-half of
one percent (1.5%) per annum.

         Section 2. Sections 4.1 through 4.4 of the Agreement are deleted and
the following Sections are substituted in lieu thereof:

                  Section 4.1. Debt to Worth Ratio. The Borrower shall maintain
         a Debt to Worth Ratio of not more than 1.85 to 1.00 as of the Fiscal
         Quarter Ending March 31, 2001; 1.75 to 1.00 for the Fiscal Quarter
         Ending June 30, 2001; 1.65 to 1.00 for the Fiscal Quarter Ending
         September 30, 2001; and 1.60 to 1.00 for the Fiscal Quarter Ending
         December 31, 2001 and the end of each Fiscal Quarter, thereafter.

                  Section 4.2. Adjusted Funded Debt to EBITDAR Ratio. While any
         of the Indebtedness is outstanding, Borrower will maintain (on a
         consolidated basis) a ratio of Adjusted Funded Debt to earnings before
         interest, taxes, depreciation, amortization and rents ("EBITDAR") of no
         greater than 2.5 to 1.0 through December 31, 2001, and 2.0 to 1.0
         thereafter, calculated on an annualized rolling four fiscal quarter
         basis




<PAGE>   2

         and measured at the end of each fiscal quarter commencing March
         31, 2001.

                  Section 4.3. Net Worth. Borrower shall maintain on a
         consolidated basis as of the end of each fiscal quarter a Net Worth of
         not less than $29,650,000 as of March 31, 2001; a net worth of
         $30,000,000 as of June 30, 2001; a net worth of $30,300,000 as of
         September 30, 2001; and a net worth of $30,500,000 as of December 31,
         2001 plus fifty percent (50%) of cumulative net income (provided that
         any net loss arising from any fiscal quarter or other accounting period
         shall be counted as zero in calculating cumulative net income) from
         that date (December 31, 2001) through subsequent fiscal quarter ends.

                  Section 4.4. Cash Flow Coverage. Borrower shall maintain (on a
         consolidated basis) as of the end of each fiscal quarter (calculated on
         a rolling four quarter basis) a Cash Flow Coverage Ratio of not less
         than 1.25 to 1.0.

         Section 3. Section 4.5 is amended to add the following definitions
thereto:

                  "Cash Flow" shall mean, in any fiscal period, the net income
         of Borrower plus depreciation plus amortization of intangible assets
         plus the interest portion of scheduled debt service plus taxes plus
         payments made under operating leases less dividends paid to
         shareholders, all on a consolidated basis and as determined in
         accordance with generally accepted accounting principles.

                  "Cash Flow Coverage Ratio" shall mean the ratio of Cash Flow
         to (a) current maturities of long-term indebtedness and interest
         payments relating thereto (including payments made pursuant to
         capitalized leases) plus (b) payments made under operating leases, all
         as determined with regard to Borrower on a consolidated basis in
         accordance with generally accepted accounting principles.

                  "Debt to Worth Ratio" shall mean the ratio of consolidated
         total liabilities of Borrower to consolidated net worth of Borrower,
         all as determined on a consolidated basis and in accordance with
         generally accepted accounting principles.

         Section 4. The following is added as Section 4.6 of the Loan Agreement:

                  4.6 Capital Expenditures and Acquisitions. Without the prior
         written consent of Lender, Borrower, and its Subsidiaries shall not
         individually or collectively make aggregate capital expenditures in any
         fiscal year in excess of $5,000,000 or make acquisitions of stock or
         assets in any fiscal year where the aggregate purchase price for such
         stock or assets is in excess of $3,000,000.



                                       2
<PAGE>   3

         Section 5. The Borrower and Lender hereby agree and acknowledge that
Borrowers' charge against earnings of approximately $9,900,000 which was made in
the fourth quarter of 2000, will not be factored into the calculations of the
financial covenants of this Loan Agreement during the fiscal year ending
December 31, 2001.

         Section 6. The Lender hereby waives compliance by the Borrower with the
financial covenants set forth in Section 4.1 through 4.4 of the Loan Agreement
in effect prior to this Amendment.

         Section 7. All other applicable provisions of the Agreement are hereby
amended to conform to the amendments as expressed in this Amendment.

         Section 8. Except as provided herein, the Agreement shall remain
unamended and shall be in full force and effect.

         Section 9. This modification shall be governed by and construed in
accordance with the laws of the State of Tennessee.

         IN WITNESS WHEREOF, the undersigned by and through their duly
authorized officers hereby execute this Amendment as of the day and date first
set forth above.

                                        LANDAIR CORPORATION


                                        By: /s/ Andrew J. Mantey
                                            --------------------------

                                        Title: Chief Financial Officer
                                               -----------------------



                                        LANDAIR TRANSPORT, INC.


                                        By: /s/ Andrew J. Mantey
                                            --------------------------

                                        Title: Chief Financial Officer
                                               -----------------------



                                        SUNTRUST BANK


                                        By:/s/ William H. Crawford
                                           ---------------------------

                                        Title: Vice President
                                               -----------------------




                                       3